Variant, recombinant beta-glucocerebrosidase proteins with increased stability and increased retained catalytic activity

ABSTRACT

Described herein are variant, recombinant β-glucocerebrosidase proteins characterized as having increased stability relative to recombinant wild-type β-glucocerebrosidase. Also provided herein are variant, recombinant β-glucocerebrosidase proteins characterized as retaining more catalytic activity relative to recombinant wild-type β-glucocerebrosidase. Further described herein are variant, recombinant β-glucocerebrosidase proteins that can have amino acid variations at one or more of the following positions: 316, 317, 321 and 145. Methods of making the variant, recombinant β-glucocerebrosidase proteins are also described as well as methods of treating patients having lysosomal storage diseases.

CROSS-REFERENCE TO RELATED APPLICATIONS

Priority is claimed under 35 U.S.C. §119(e) to provisional U.S.Provisional Application Ser. No. 61/411,331, filed Nov. 8, 2010, andU.S. Provisional Application Ser. No. 61/412,180, filed Nov. 10, 2010,both of which are incorporated herein by reference and in theirentireties.

TECHNICAL FIELD

The field of this invention includes proteins that are useful in enzymereplacement therapy for various lysosomal storage diseases. Theselysosomal storage diseases include Gaucher disease.

BACKGROUND

β-glucocerebrosidase is a soluble lysosomal enzyme that functions at theluminal membrane surface through interactions with Saposin C and anionicphospholipids to hydrolyze the glycolipid glucosylceramide.β-glucocerebrosidase is particularly important in tissue macrophages tobreak down and recycle membranes from engulfed damaged cells andpathogens. Since glucosylceramide is the primary precursor molecule formore than 300 μlycolipids and gangliosides that are involved in numerousimportant cellular pathways and signaling cascades, it is essential tomaintain the intricate balance for these various lipid molecules.

Gaucher disease is caused by a deficiency in β-glucocerebrosidase thatresults in the accumulation of glucosylceramide. Gaucher diseasemanifests itself through various clinical symptoms including anemia,thrombocytopenia, hepatosplenomegaly and skeletal abnormalities. Gaucherdisease is classified in three categories based on neurologicalinvolvement: type 1 (non-neuronopathic); type 2 (acute neuronopathic);and type 3 (chronic neuronopathic). There is no known cure for Gaucherdisease, but enzyme replacement therapy (ERT), which supplements thedeficient β-glucocerebrosidase and substrate reduction therapy whichinhibits the synthesis of glucosylceramide are approved treatments forthis disease. Other therapeutic approaches such as small moleculepharmacological chaperones and protein folding modulators are also beingevaluated as potential treatments of this disease. Of these treatmentapproaches, ERT is the most established and effective clinical treatmentfor the visceral symptoms of Gaucher disease. Imiglucerase (recombinantβ-glucocerebrosidase; Cerezyme™, Genzyme Corp.™) was developed andapproved by the FDA in 1994 for the treatment of Gaucher disease and iscurrently the standard of care for this disease.

While Cerezyme™ ERT is widely considered to be the most effectivetreatment, this lysosomal enzyme is not stable at neutral pH and 37° C.In fact, the vast majority of the drug is irreversibly inactivated inblood shortly after intravenous infusion. Only the small fraction thatretains catalytic activity and is internalized into the targetmacrophages confers the entire therapeutic effect. Hence, it would beadvantageous to develop a more stable β-glucocerebrosidase enzyme thatis not as susceptible to enzyme inactivation from the protein productionstep through physiological conditions that would be encountered uponintroduction into a human subject in need thereof.

SUMMARY

Provided herein are variant, recombinant β-glucocerebrosidase proteinscharacterized as having increased stability relative to wild-type,recombinant β-glucocerebrosidase. Also provided herein are variant,recombinant β-glucocerebrosidase proteins characterized as retainingmore catalytic activity relative to wild-type, recombinantβ-glucocerebrosidase. Further described herein are variant, recombinantβ-glucocerebrosidase proteins that can have amino acid variations at oneor more of the following positions: 316, 317, 321 and 145.

Described herein are methods of making the variant, recombinantβ-glucocerebrosidase proteins characterized as retaining more catalyticactivity relative to wild-type, recombinant β-glucocerebrosidase.Further described herein are compositions comprising the variant,recombinant β-glucocerebrosidase proteins and a pharmaceuticallyacceptable carrier. Also provided herein are compositions comprising thevariant, recombinant β-glucocerebrosidase proteins and apharmaceutically acceptable buffer.

Described herein are variant, recombinant β-glucocerebrosidase proteinshaving one or more replacement amino acids in the loop1 region of theprotein, the one or more replacement amino acids characterized as havinga side-chain conformation that increases order near the active site ofthe protein. Further described herein are variant, recombinantβ-glucocerebrosidase proteins having one or more replacement amino acidsin an α-helix near the active site (α6) of the protein, the one or morereplacement amino acid side-chain characterized as having a side-chainconfirmation that stabilizes this helix and pulls adjacent loop1 awayfrom the catalytic site and has an open and active conformation. Alsoprovided herein are variant, recombinant β-glucocerebrosidase proteinshaving one or more replacement amino acids in the random coil regionbetween beta-sheet (β2) and an α-helix (α2), the one or more replacementamino acid side-chains characterized as having a side-chain confirmationthat facilitates better interactions between different residues andsecondary structures for improved stability.

Provided herein are methods for treating a lysosomal storage disease byadministering to a subject in need thereof a variant, recombinantβ-glucocerebrosidase protein characterized as having increased stabilitycompared to wild-type, recombinant β-glucocerebrosidase. Also providedherein are methods for treating a lysosomal storage disease byadministering to a subject in need thereof a variant, recombinantβ-glucocerebrosidase protein characterized as retaining increasedcatalytic activity compared to wild-type, recombinantβ-glucocerebrosidase. Also provided herein are methods for treating alysosomal storage disease by administering to a subject in need thereofa variant, recombinant β-glucocerebrosidase protein characterized ashaving an increased specific activity compared to wild-type, recombinantβ-glucocerebrosidase. Also provided herein are methods for treating alysosomal storage disease by administering to a subject in need thereofa variant, recombinant β-glucocerebrosidase protein having an amino acidvariation at one or more of the following positions: 316, 317, 321, and145. Also provided herein are methods for treating Gaucher disease byadministering to a subject in need thereof a variant, recombinantβ-glucocerebrosidase protein.

Also provided herein are compounds comprising a variant, recombinantβ-glucocerebrosidase protein characterized as having any one of thefollowing amino acid sequences: SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO:4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9,SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO:14, SEQ ID NO: 15, or SEQ ID NO: 16.

Also provided herein are variant, recombinant β-glucocerebrosidaseproteins characterized as capable of increased expression relative towild-type, recombinant β-glucocerebrosidase.

Also provided herein are methods of making a compound comprising avariant, recombinant β-glucocerebrosidase protein characterized ashaving any one of the following amino acid sequences: SEQ ID NO: 2, SEQID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ IDNO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ IDNO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16. Also providedherein are methods of making a nucleic acid encoding a variant,recombinant β-glucocerebrosidase protein characterized as having any oneof the following amino acid sequences: SEQ ID NO: 2, SEQ ID NO: 3, SEQID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ IDNO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other aspects of the present invention is apparentfrom the following detailed description of the invention when consideredin conjunction with the accompanying drawings. For the purpose ofillustrating the invention, there is shown in the drawings embodimentsthat are presently preferred, it being understood, however, that theinvention is not limited to the specific instrumentalities disclosed.The drawings are not necessarily drawn to scale. In the drawings:

FIG. 1 (A) shows enzyme activity measurements to compare the relativeexpression of variant, recombinant β-glucocerebrosidase proteins to thewild-type enzyme secreted into cell culture medium from a typicaltransient transfection experiment after 48 hrs; (B) shows a western blotanalysis to compare the relative amounts of secreted variant,recombinant β-glucocerebrosidase proteins and the wild-type GlcCeraseprotein in cell culture medium after transient transfection.

FIG. 2 shows the stability of variant, recombinant β-glucocerebrosidaseproteins with the H145L modification, the H145F modification, theF316A/L317F modifications, the K321N modification, the K321Amodification, the F316A/L317F/K321N modifications, and the H145L/K321Nmodifications compared to wild-type, recombinant β-glucocerebrosidaseprotein at pH 7.5 and 37° C.; and

FIG. 3 shows the stability of the variant, recombinantβ-glucocerebrosidase protein with the F316A/L317F modifications comparedto wild-type, recombinant β-glucocerebrosidase protein at pH 8 and 37°C.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The present subject matter may be understood more readily by referenceto the following detailed description taken in connection with theaccompanying figures and examples, which form a part of this disclosure.It is to be understood that this invention is not limited to thespecific devices, methods, applications, conditions or parametersdescribed and/or shown herein, and that the terminology used herein isfor the purpose of describing particular embodiments by way of exampleonly and is not intended to be limiting of the claimed invention.

Also, as used in the specification including the appended claims, thesingular forms “a,” “an,” and “the” include the plural, and reference toa particular numerical value includes at least that particular value,unless the context clearly dictates otherwise. The term “plurality”, asused herein, means more than one. When a range of values is expressed,another embodiment includes from the one particular value and/or to theother particular value. Similarly, when values are expressed asapproximations, by use of the antecedent “about,” it is understood thatthe particular value forms another embodiment. All ranges are inclusiveand combinable.

Examples are provided to assist in a further understanding of theinventions. Particular materials used, protocols and conditions areintended to be further illustrative of the inventions and should not beconstrued to limit the reasonable scope thereof.

Unless noted differently, variant, recombinant β-glucocerebrosidaseprotein properties are stated relative to wild-type, recombinantβ-glucocerebrosidase protein properties (SEQ ID NO: 1). Herein,“GlcCerase” is an abbreviation used for β-glucocerebrosidase. All aminoacid numbers are relative to SEQ. ID. NO. 1. Thus, position 145 would bethe 145th amino acid occurring in SEQ. ID. NO. 1. Furthermore, variant,recombinant β-glucocerebrosidase proteins as disclosed herein alsoinclude functional fragments or derivatives thereof.

Herein, “about neutral pH” is meant to include the pHs that are normallyconsidered physiological pHs (i.e., a pH of about 7.5 at 37° C.).

Suitable variant, recombinant β-glucocerebrosidase proteins can becharacterized as having similar or increased protein expression andsecretion into cell culture medium relative to wild-type, recombinantβ-glucocerebrosidase proteins during cell culture and proteinproduction. Suitable variant, recombinant β-glucocerebrosidase proteinscan be characterized as having increased stability relative towild-type, recombinant β-glucocerebrosidase proteins. These proteinshave increased stability at physiological conditions and thosephysiological conditions can be in vivo. Further, these proteins canalso be characterized as having increased stability at conditions ofabout neutral pH and about 37° C. This increased stability can bemonitored at conditions of about neutral pH and about 37° C. over aperiod of about three hours. The increased stability is retained in cellculture medium and can be retained inside cells. These proteins can alsohave increased stability inside the lysosome and inside the lysosome theconditions can be about pH 5 and about 37° C. These proteins can alsohave increased stability that is characterized by reduced proteolyticdegradation and the reduced proteolytic degradation can occur in cells.Further, the reduced proteolytic degradation can be in the lysosome inthe cells. These proteins can also have increased stability atnon-physiological conditions such as in buffer solutions during proteinpurification. These buffer solutions can have a pH greater than about 7or a pH less than about 3 during protein purification. These buffersolutions can also contain organic solvents or chaotropic salts. Theseproteins can also have increased stability in buffer solutions attemperatures that range from about 2° C. to about 37° C. Further, theseproteins can also have increased stability in buffer solutions attemperatures that range from about 2° C. to about 8° C. Also, theseproteins can have increased stability in buffer solutions at atemperature about 20° C. These proteins can also have increasedstability after freeze-thaw cycles or reconstitution afterlyophilization. These proteins can also have increased stability in adrug-formulation buffer such as saline.

Suitable variant, recombinant β-glucocerebrosidase proteins, can becharacterized as retaining more catalytic activity relative towild-type, recombinant β-glucocerebrosidase. These proteins haveretained more catalytic activity at physiological conditions and themore retained catalytic activity can be in vivo. Further, these proteinscan also be characterized as having retained more catalytic activity atconditions of about neutral pH and about 37° C. This more retainedcatalytic activity can be monitored at conditions of about neutral pHand about 37° C. over a period of about two hours. These proteins haveretained more catalytic activity in cell culture medium and the moreretained catalytic activity can be inside cells. Further, these proteinscan have retained more catalytic activity inside the lysosome and insidethe lysosome the conditions can be about pH 5 and about 37° C. Theseproteins can also have retained more catalytic activity that ischaracterized by reduced proteolytic degradation and the reducedproteolytic degradation can occur in cells. Further, the reducedproteolytic degradation can be in the lysosome in the cells. Theseproteins can also have retained more catalytic activity atnon-physiological conditions such as in buffer solutions during proteinpurification. These buffer solutions can have a pH greater than about 7or a pH less than about 3 during protein purification. These buffersolutions can also contain organic solvents or chaotropic salts. Theseproteins can also have retained more catalytic activity at temperaturesthat range from about 2° C. to about 37° C. Further, these proteins canalso have retained more catalytic activity at temperatures that rangefrom about 2° C. to about 8° C. Also, these proteins can have retainedmore catalytic activity at a temperature of about 20° C. These proteinscan also have retained more catalytic activity after freeze-thaw cyclesor reconstitution after lyophilization. These proteins can also haveretained more catalytic activity in a drug-formulation buffer such assaline.

Variant, recombinant β-glucocerebrosidase proteins can also have anamino acid variation at one or more of the following positions: 316,317, 321 and 145. These proteins can have the amino acid variation orvariations: (1) F316A and L317F; or (2) K321N; or (3) H145L; or (4)H145F; or (5) F316A, L317F, and K321N; or (6) K321A; or (7) K321V; (8)F316A, L317F, and K321A; or (9) F316A, L317F, and K321V; or (10) H145L,F316A, and L317F; or (11) H145L and K321N; or (12) H145L and K321A; or(13) H145L and K321V. Some of these proteins can also be characterizedas having similar or increased protein expression and secretion intocell culture medium relative to wild-type, recombinantβ-glucocerebrosidase proteins during cell culture and proteinproduction. These proteins can also be characterized as having increasedstability relative to wild-type, recombinant β-glucocerebrosidase. Theseproteins can also be characterized as having increased stability atphysiological conditions and these conditions are about neutral pH andabout 37° C. These proteins can also be characterized as retaining morecatalytic activity relative to wild-type, recombinantβ-glucocerebrosidase. The catalytic activity can be measured afterincubation at conditions of about neutral pH and about 37° C. over aperiod of about two hours. These proteins can also be characterized asretaining more catalytic activity at physiological conditions. Thevariant, recombinant β-glucocerebrosidase protein with the amino acidvariations F316A and L317F has an extended apparent half-life of atleast about 2-fold longer than wild-type, recombinantβ-glucocerebrosidase. The variant, recombinant β-glucocerebrosidaseprotein with the amino acid variation K321N has an extended apparenthalf-life of at least about 3-fold longer than wild-type, recombinantβ-glucocerebrosidase. The variant, recombinant β-glucocerebrosidaseprotein with the amino acid variation K321A has an extended apparenthalf-life of at least about 3-fold longer than wild-type, recombinantβ-glucocerebrosidase. The variant, recombinant β-glucocerebrosidaseprotein with the amino acid variation K321V has an extended apparenthalf-life of at least about 1.4-fold longer than wild-type, recombinantβ-glucocerebrosidase. The variant, recombinant β-glucocerebrosidaseprotein with the amino acid variation H145L has an extended apparenthalf-life of at least about 3-fold longer than wild-type, recombinantβ-glucocerebrosidase. The variant, recombinant β-glucocerebrosidaseprotein with the amino acid variation H145F has an extended apparenthalf-life of at least about 2-fold longer than wild-type, recombinantβ-glucocerebrosidase. The variant, recombinant β-glucocerebrosidaseprotein with the amino acid variations F316A, L317F, and K321N has anextended apparent half-life of at least about 3-fold longer thanwild-type, recombinant β-glucocerebrosidase. The variant, recombinantβ-glucocerebrosidase protein with the amino acid variations H145L,F316A, and L317F has an extended apparent half-life of at least about2-fold longer than wild-type, recombinant β-glucocerebrosidase. Thevariant, recombinant β-glucocerebrosidase protein with the amino acidvariations H145L and K321N has an extended apparent half-life of atleast about 3-fold longer than wild-type, recombinantβ-glucocerebrosidase. This extended apparent half-life can be measuredafter incubation at conditions of about neutral pH and about 37° C. overa period of about two hours.

Example 1 Reagents

4-methylumbelliferyl-β-D-glucoside (4MUG) fluorogenic substrate waspurchased from Research Products International (Mt. Prospect, Ill.). DNAGel Extraction and Miniprep DNA® kits were from QIAGEN® (Valencia,Calif.). PureYield Maxiprep DNA Kit™ was from Promega™ (Madison, Wis.).Unless stated otherwise, chemicals were from Sigma™ (St. Louis, Mo.).Fugene-HD™ transfection reagent was from Roche™ (Indianapolis, Ind.).pEF6/V5-HisA™ mammalian expression vector, Dulbecco's modified Eaglemedium (DMEM), fetal bovine serum (FBS) and other tissue culturereagents were from Invitrogen™ (Carlsbad, Calif.). Wild-type humanwild-type β-glucocerebrosidase cDNA (NM_(—)000157.3) was purchased fromOrigene™ (Rockville, Md.). Restriction endonucleases, Phusion-HF™ DNApolymerase, T4 DNA ligase, Antarctic phosphatase, chemically-competentE. coli (DH5a cells) and endoglycosidases PNGaseF and EndoH were allpurchased from New England Biolabs™ (Ipswich, Mass.). Human embryonickidney cells (transformed with the T-antigen; HEK293T) was from ATCC™.

Assays

Variant, recombinant β-glucocerebrosidase proteins and wild-type,recombinant β-glucocerebrosidase protein were all tested by transientexpression in a human cell line (HEK293T) to assess protein expression.The reporter system to test catalytic activity of the variant,recombinant β-glucocerebrosidase proteins or wild-type, recombinantβ-glucocerebrosidase protein was the ability to hydrolyze the4-MU-β-glucose fluorogenic substrate at about pH 5.2 and about 37° C.The stability of these variant, recombinant β-glucocerebrosidaseproteins and the purified wild-type, recombinant β-glucocerebrosidaseprotein was tested by monitoring of the retention of catalytic activityof each protein after incubation at about neutral pH and about 37° C.over a period of about 3 hours. The conditions in these experiments weredesigned to resemble the environment that would exist during theintravenous infusion of β-glucocerebrosidase protein enzyme replacementtherapy into a patient.

More specifically but in no way to be construed as limiting, HEK293Tcells were plated in 12-well tissue culture plates with 1 ml of DMEMmedium supplemented with 10% FBS and incubated at 37° C. with a 5% CO₂atmosphere. When the HEK293T cells reached 80-90% confluency, the spentmedium was replaced with 1 ml of fresh DMEM/10% FBS medium and each wellwas transfected with 1 μg plasmid DNA for individualβ-glucocerebrosidase proteins or PBS (for a mock-transfected negativecontrol) and 3 μl of Fugene-HD transfection reagent according to themanufacturer's protocol. Transfected cells were incubated for 24-72hours and checked daily for expression of recombinantβ-glucocerebrosidase protein (secreted into medium) via enzyme activityassays.

β-glucocerebrosidase protein expression (and secretion into cell culturemedium) was assessed by enzyme activity assays using conditioned mediumfrom transient transfection experiments after 24, 48 or 72-hrs and the4-methylumbelliferyl-β-D-glucoside (4-MUG) fluorogenic substrate.Briefly, 20 μl of conditioned media from each sample was harvested atthe indicated time points and diluted with 80 μl Mcllvane buffer (MIbuffer: 50 mM sodium citrate/sodium phosphate (pH 5.2)/0.25% (v/v)Triton X-100/0.25% (w/v) sodium taurocholate) in 0.5 ml microcentrifugetubes. Twenty five μl of each diluted sample was aliquotted intoindividual wells of 96-well black clear bottom plates (performed intriplicate) and 50 μl of 6 mM 4-MUG substrate (prepared in MI buffer)was added to each well via a multi-channel pipettor. The plates werethen sealed with cover tape and incubated at 37° C. for 1 hr. Theenzymatic reactions were halted by adding 125 μl of 0.1 M NaOH and theliberated 4-MU fluorescence was read on a fluorescence plate readerusing 355 nm excitation and 460 nm emission wavelengths, respectively.The 4-MU fluorescence from the mock-transfected sample served as the“background” control and subtracted from all β-glucocerebrosidaseprotein samples.

To estimate the amount of wild-type and variant acidβ-glucocerebrosidase proteins present in cell culture medium,conditioned cell culture media from transient transfection experimentswere subjected to sodium dodecylsulfate polyacrylamide gelelectrophoresis (SDS-PAGE) and transferred to nitrocellulose membraneusing standard techniques. The membrane was then incubated with BlockingBuffer: 4% (w/v) non-fat milk in 50 mM TRIS-buffered saline (pH7.5)/0.1% (v/v) Tween-20 (TBST) for 1 hr at room temperature withshaking. The membrane was subsequently incubated with rabbit anti-humanβ-glucocerebrosidase polyclonal primary antibodies (raised against a 19amino acid peptide corresponding to C-terminus of human acidβ-glucocerebrosidase; Sigma G-4171) diluted 1:2500 in Blocking Bufferfor 1 hr at room temp or overnight at 4° C. with shaking The blot wasthen washed with TBST at room temp with shaking and at least threebuffer changes over 1 hr. The blot was then incubated with anenzyme-linked secondary antibody (e.g., horseradishperoxidase-conjugated goat anti-rabbit antibodies) diluted 1:10,000 inBlocking Buffer for 1 hr at room temp with shaking The blot was washedwith TBST at room temp with shaking and at least three buffer changesover 1 hr. The blot was then incubated with chemiluminescence substrate(e.g., Pierce's Supersignal West-Dura Extended Duration Substrate™) for5 min at room temp and visualized by an imaging system or by film toassess the level of protein expression for wild-type and variant acidβ-glucocerebrosidase enzymes.

To assess β-glucocerebrosidase protein stability, the transientlyexpressed variant, recombinant β-glucocerebrosidase proteins and thepurified wild-type, recombinant β-glucocerebrosidase protein (i.e.Cerezyme™) were incubated in neutral pH buffer at 37° C. and assayed forenzyme activity to determine the extent of loss of catalytic activityover a period of about three hours. Briefly, 60 μl of conditioned medium(containing individual variant, recombinant β-glucocerebrosidaseproteins) was harvested after 24 or 48 hrs post-transfection and addedto 420 μl of 0.1 M potassium phosphate (pH 7.5)/0.2% (v/v) TritonX-100/0.2% (w/v) BSA in 0.5 ml microfuge tubes. Similarly, purifiedwild-type, recombinant β-glucocerebrosidase protein was serially dilutedto 1:12,500 in DMEM medium/10% FBS and 60 μl of this diluted wild-type,recombinant β-glucocerebrosidase protein sample was added to 420 μl 0.1M potassium phosphate (pH 7.5)/0.2% (v/v) Triton X-100/0.2% (w/v) BSA toobtain a final dilution of 1:100,000. (This amount of wild-type,recombinant β-glucocerebrosidase protein was empirically determined tocontain similar amounts of enzymatic activity as transiently expressedrecombinant β-glucocerebrosidase proteins). All samples were incubatedin a 37° C. waterbath and 70 μl of each sample was removed at specifiedtime points (0, 30, 60, 90, 120 or 180 min) and added to new 0.5 mlmicrocentrifuge tubes containing 20 μl of 0.5 M NaOAc (pH 5.2). Thesamples were mixed thoroughly and 25 μl of each diluted sample was usedto measure the residual recombinant β-glucocerebrosidase protein enzymeactivity (in triplicate) as described above. The initial enzyme activity(at t=0 min) was designated as 100% for each recombinantβ-glucocerebrosidase protein while the enzymatic activities for allsubsequent time points were normalized to the initial activity todetermine the residual enzyme activity over the entire time course. Datafrom multiple experiments (at least 2 separate experiments) were used toobtain average values for residual recombinant β-glucocerebrosidaseprotein activity for individual time points and graphed relative toincubation time as shown.

Example 2

The expression of variant, recombinant β-glucocerebrosidase proteinswith specific amino acid substitutions were compared to wild-type,recombinant β-glucocerebrosidase protein (“wild-type GlcCerase”) usingtransient transfection experiments described above (FIG. 1). As can beseen in FIG. 1A, the variant, recombinant β-glucocerebrosidase proteinwith the F316A and L317F variations (“GlcCerase-F316A/L317F”), thevariant, recombinant β-glucocerebrosidase protein with the K321Nvariation (“GlcCerase-K321N”), the variant, recombinantβ-glucocerebrosidase protein with the H145L variation(“GlcCerase-H145L”), the variant, recombinant β-glucocerebrosidaseproteins with the F316A, L317F and K321N variations(“GlcCerase-F316A/L317F/K321N”), the variant, recombinantβ-glucocerebrosidase proteins with the H145L and K321N variations(“GlcCerase-H145L/K321N”) were transiently expressed with the wild-typeGlcCerase in HEK293T cells. The conditioned cell culture medium washarvested 48 hrs after transfection and assayed for β-glucocerebrosidaseenzyme activity using the 4-methylumbelliferyl-β-D-glucoside (4-MUG)fluorogenic substrate to assess the relative level of expression ofthese variant GlcCerase enzymes compared to wild-type GlcCerase. Theseresults show that different variant GlcCerase enzymes were expressed aswell or better than wild-type GlcCerase as evidenced by the highermeasured enzyme activity in cell culture medium. Other variant GlcCeraseenzymes including GlcCerase-H145F, GlcCerase-H145L/F316A/L317F/K321Nwere also expressed better than wild-type GlcCerase (data not shown).GlcCerase-K321A and GlcCerase-H145F/F316A/L317F/K321N were expressed atapproximately the same level as wild-type GlcCerase whileGlcCerase-K321V was expressed less efficiently than wild-type GlcCerase(data not shown).

The amount of variant GlcCerase proteins and wild-type GlcCerase proteinpresent in conditioned cell culture medium was evaluated using Westernblotting as described above. As can be seen in FIG. 1B, higher amountsof GlcCerase-F316A/L317F and GlcCerase-K321N proteins were present inconditioned cell culture medium than wild-type GlcCerase. These Westernblotting data are consistent with the GlcCerase enzyme activity resultsand confirm that certain variant GlcCerase enzymes are expressed andsecreted better than wild-type GlcCerase. Similar results were observedfor other variant GlcCerase proteins (data not shown).

Example 3

The variant, recombinant β-glucocerebrosidase protein with the F316A andL317F variations (“GlcCerase-F316A/L317F”) was compared to purifiedwild-type, recombinant β-glucocerebrosidase protein (“wild-typeGlcCerase”) using the in vitro stability assay described above. Theconditions used for the variant and wild-type proteins were identical.As can be seen in FIG. 2, GlcCerase-F316A/L317F is significantly morestable than wild-type GlcCerase at about neutral pH and about 37° C.over a two or three hour time course. GlcCerase-F316A/L317F retained 85%of its initial activity after 30 minutes of incubation.GlcCerase-F316A/L317F retained 73% of its initial activity after 60minutes of incubation. GlcCerase-F316A/L317F retained 50% of its initialactivity after 120 minutes of incubation. GlcCerase-F316A/L317F retained34% of its initial activity after 180 minutes of incubation. Bycomparison, wild-type GlcCerase treated under the same experimentalconditions retained 68% of its initial activity after 30 minutes ofincubation. Wild-type GlcCerase retained 46% of its initial activityafter 60 minutes of incubation. Wild-type GlcCerase retained 20% of itsinitial activity after 120 minutes of incubation. Wild-type GlcCeraseretained 8% of its initial activity after 180 minutes of incubation.

The estimated half-life (operationally defined in this study as theincubation time at about pH 7.5 and about 37° C. that resulted in a 50%loss of initial enzyme activity) for GlcCerase-F316A/L317F isapproximately 120 minutes while the estimated half-life for wild-typeGlcCerase is approximately 50 minutes under these experimentalconditions. GlcCerase-F316A/L317F was also tested at about pH 8 andabout 37° C. and displayed a similar trend (FIG. 3). BothGlcCerase-F316A/L317F and wild-type GlcCerase were less stable at aboutpH 8 and about 37° C., but these results confirm thatGlcCerase-F316A/L317F is more stable at higher pH than wild-typeGlcCerase.

Example 4

The variant, recombinant β-glucocerebrosidase protein with the K321Nvariation (“GlcCerase-K321N”) was compared to purified wild-type,recombinant β-glucocerebrosidase protein (“wild-type GlcCerase”) usingthe assay described above. The conditions used for the variant andwild-type proteins were identical. As can be seen in FIG. 2,GlcCerase-K321N is significantly more stable than wild-type GlcCerase atabout neutral pH and about 37° C. over a three hour time course.GlcCerase-K321N retained 104% of its initial activity after 30 minutesof incubation. GlcCerase-K321N retained 91% of its initial activityafter 60 minutes of incubation. GlcCerase-K321N retained 76% of itsinitial activity after 120 minutes of incubation. GlcCerase-K321Nretained 54% of its initial activity after 180 minutes of incubation. Bycomparison, wild-type GlcCerase treated under the same experimentalconditions retained 68% of its initial activity after 30 minutes ofincubation. Wild-type GlcCerase retained 46% of its initial activityafter 60 minutes of incubation. Wild-type GlcCerase retained 20% of itsinitial activity after 120 minutes of incubation. Wild-type GlcCeraseretained 8% of its initial activity after 180 minutes of incubation.

The estimated half-life (operationally defined in this study as theincubation time at about pH 7.5 and about 37° C. that resulted in a 50%loss of initial enzyme activity) for GlcCerase-K321N is approximately180 minutes while the estimated half-life for wild-type GlcCerase isapproximately 50 minutes under these experimental conditions.

Example 5

The variant, recombinant β-glucocerebrosidase protein with the H145Lvariation (“GlcCerase-H145L”) was compared to purified wild-type,recombinant β-glucocerebrosidase protein (“wild-type GlcCerase”) usingthe assay described above. The conditions used for the variant andwild-type proteins were identical. As can be seen in FIG. 2,GlcCerase-H145L is significantly more stable than wild-type GlcCerase atabout neutral pH and about 37° C. over a three hour time course.GlcCerase-H145L retained 92% of its initial activity after 30 minutes ofincubation. GlcCerase-H145L retained 87% of its initial activity after60 minutes of incubation. GlcCerase-H145L retained 62% of its initialactivity after 120 minutes of incubation. GlcCerase-H145L retained 42%of its initial activity after 180 minutes of incubation. By comparison,wild-type GlcCerase treated under the same experimental conditionsretained 68% of its initial activity after 30 minutes of incubation.Wild-type GlcCerase retained 46% of its initial activity after 60minutes of incubation. Wild-type GlcCerase retained 20% of its initialactivity after 120 minutes of incubation. Wild-type GlcCerase retained8% of its initial activity after 180 minutes of incubation.

The estimated half-life (operationally defined in this study as theincubation time at about pH 7.5 and about 37° C. that resulted in a 50%loss of initial enzyme activity) for GlcCerase-H145L is approximately160 minutes while the estimated half-life for wild-type GlcCerase isapproximately 50 minutes under these experimental conditions.

Example 6

The variant, recombinant β-glucocerebrosidase protein with the H145Fvariation (“GlcCerase-H145F”) was compared to purified wild-type,recombinant β-glucocerebrosidase protein (“wild-type GlcCerase”) usingthe assay described above. The conditions used for the variant andwild-type proteins were identical. As can be seen in FIG. 1,GlcCerase-H145F is significantly more stable than wild-type GlcCerase atabout neutral pH and about 37° C. over a three hour time course.GlcCerase-H145F retained 94% of its initial activity after 30 minutes ofincubation. GlcCerase-H145F retained 80% of its initial activity after60 minutes of incubation. GlcCerase-H145F retained 37% of its initialactivity after 120 minutes of incubation. GlcCerase-H145F retained 17%of its initial activity after 180 minutes of incubation. By comparison,wild-type GlcCerase treated under the same experimental conditionsretained 68% of its initial activity after 30 minutes of incubation.Wild-type GlcCerase retained 46% of its initial activity after 60minutes of incubation. Wild-type GlcCerase retained 20% of its initialactivity after 120 minutes of incubation. Wild-type GlcCerase retained8% of its initial activity after 180 minutes of incubation.

The estimated half-life (operationally defined in this study as theincubation time at about pH 7.5 and about 37° C. that resulted in a 50%loss of initial enzyme activity) for GlcCerase-H145F is approximately100 minutes while the estimated half-life for wild-type GlcCerase isapproximately 50 minutes under these experimental conditions.

Example 7

The variant, recombinant β-glucocerebrosidase protein with the F316A,L317F, and K321N variations (“GlcCerase-F316A/L317F/K321N”) was comparedto purified wild-type, recombinant β-glucocerebrosidase protein(“wild-type GlcCerase”) using the assay described above. The conditionsused for the variant and wild-type proteins were identical. As can beseen in FIG. 2, GlcCerase-F316A/L317F/K321N is significantly more stablethan wild-type GlcCerase at about neutral pH and about 37° C. over athree hour time course. GlcCerase-F316A/L317F/K321N retained 101% of itsinitial activity after 30 minutes of incubation.GlcCerase-F316A/L317F/K321N retained 91% of its initial activity after60 minutes of incubation. GlcCerase-F316A/L317F/K321N retained 75% ofits initial activity after 120 minutes of incubation.GlcCerase-F316A/L317F/K321N retained 52% of its initial activity after180 minutes of incubation. By comparison, wild-type GlcCerase treatedunder the same experimental conditions retained 68% of its initialactivity after 30 minutes of incubation. Wild-type GlcCerase retained46% of its initial activity after 60 minutes of incubation. Wild-typeGlcCerase retained 20% of its initial activity after 120 minutes ofincubation. Wild-type GlcCerase retained 8% of its initial activityafter 180 minutes of incubation.

The estimated half-life (operationally defined in this study as theincubation time at about pH 7.5 and about 37° C. that resulted in a 50%loss of initial enzyme activity) for GlcCerase-F316A/L317F/K321N isapproximately 180 minutes while the estimated half-life for wild-typeGlcCerase is approximately 50 minutes under these experimentalconditions.

Example 8

The variant, recombinant β-glucocerebrosidase protein with the H145L,F316A and L317F variations (“GlcCerase-H145L/F316A/L317F”) was comparedto purified wild-type, recombinant β-glucocerebrosidase protein(“wild-type GlcCerase”) using the assay described above. The conditionsused for the variant and wild-type proteins were identical.GlcCerase-H145L/F316A/L317F is significantly more stable than wild-typeGlcCerase at about neutral pH and about 37° C. over a three hour timecourse. GlcCerase-H145L/F316A/L317F retained approximately 77% of itsinitial activity after 30 minutes of incubation.GlcCerase-H145L/F316A/L317F N retained 80% of its initial activity after60 minutes of incubation. GlcCerase-H145L/F316A/L317F retained 56% ofits initial activity after 120 minutes of incubation.GlcCerase-H145L/F316A/L317F retained 39% of its initial activity after180 minutes of incubation. By comparison, wild-type GlcCerase treatedunder the same experimental conditions retained 68% of its initialactivity after 30 minutes of incubation. Wild-type GlcCerase retained46% of its initial activity after 60 minutes of incubation. Wild-typeGlcCerase retained 20% of its initial activity after 120 minutes ofincubation. Wild-type GlcCerase retained 8% of its initial activityafter 180 minutes of incubation.

The estimated half-life (operationally defined in this study as theincubation time at about pH 7.5 and about 37° C. that resulted in a 50%loss of initial enzyme activity) for GlcCerase-H145L/F316A/L317F isapproximately 135 minutes while the estimated half-life for wild-typeGlcCerase is approximately 50 minutes under these experimentalconditions.

Example 9

The variant, recombinant β-glucocerebrosidase protein with the H145L andK321N variations (“GlcCerase-H145L/K321N”) was compared to purifiedwild-type, recombinant β-glucocerebrosidase protein (“wild-typeGlcCerase”) using the assay described above. The conditions used for thevariant and wild-type proteins were identical. As can be seen in FIG. 2,GlcCerase-H145L/K321N is significantly more stable than wild-typeGlcCerase at about neutral pH and about 37° C. over a three hour timecourse. GlcCerase-H145L/K321N retained 89% of its initial activity after30 minutes of incubation. GlcCerase-H145L/K321N retained 86% of itsinitial activity after 60 minutes of incubation. GlcCerase-H145L/K321Nretained 76% of its initial activity after 120 minutes of incubation.GlcCerase-H145L/K321N retained 62% of its initial activity after 180minutes of incubation. By comparison, wild-type GlcCerase treated underthe same experimental conditions retained 68% of its initial activityafter 30 minutes of incubation. Wild-type GlcCerase retained 46% of itsinitial activity after 60 minutes of incubation. Wild-type GlcCeraseretained 20% of its initial activity after 120 minutes of incubation.Wild-type GlcCerase retained 8% of its initial activity after 180minutes of incubation.

The estimated half-life (operationally defined in this study as theincubation time at about pH 7.5 and about 37° C. that resulted in a 50%loss of initial enzyme activity) for GlcCerase-H145L/K321N isapproximately 180 minutes while the estimated half-life for wild-typeGlcCerase is approximately 50 minutes under these experimentalconditions.

Example 10

The variant, recombinant β-glucocerebrosidase protein with the K321Avariation (“GlcCerase-K321A”) was compared to purified wild-type,recombinant β-glucocerebrosidase protein (“wild-type GlcCerase”) usingthe assay described above. The conditions used for the variant andwild-type proteins were identical. As can be seen in FIG. 2,GlcCerase-K321A is significantly more stable than wild-type GlcCerase atabout neutral pH and about 37° C. over a three hour time course.GlcCerase-K321A retained 90% of its initial activity after 30 minutes ofincubation. GlcCerase-K321A retained 89% of its initial activity after60 minutes of incubation. GlcCerase-K321A retained 68% of its initialactivity after 120 minutes of incubation. GlcCerase-K321A retained 49%of its initial activity after 180 minutes of incubation. By comparison,wild-type GlcCerase treated under the same experimental conditionsretained 68% of its initial activity after 30 minutes of incubation.Wild-type GlcCerase retained 46% of its initial activity after 60minutes of incubation. Wild-type GlcCerase retained 20% of its initialactivity after 120 minutes of incubation. Wild-type GlcCerase retained8% of its initial activity after 180 minutes of incubation.

The estimated half-life (operationally defined in this study as theincubation time at about pH 7.5 and about 37° C. that resulted in a 50%loss of initial enzyme activity) for GlcCerase-K321A is approximately170 minutes while the estimated half-life for wild-type GlcCerase isapproximately 50 minutes under these experimental conditions.

Example 11

The variant, recombinant β-glucocerebrosidase protein with the K321Avariation (“GlcCerase-K321V”) was compared to purified wild-type,recombinant β-glucocerebrosidase protein (“wild-type GlcCerase”) usingthe assay described above. The conditions used for the variant andwild-type proteins were identical. GlcCerase-K321V is significantly morestable than wild-type GlcCerase at about neutral pH and about 37° C.over a three hour time course. GlcCerase-K321V retained 74% of itsinitial activity after 30 minutes of incubation. GlcCerase-K321Vretained 55% of its initial activity after 60 minutes of incubation.GlcCerase-K321V retained 29% of its initial activity after 120 minutesof incubation. GlcCerase-K321V retained 15% of its initial activityafter 180 minutes of incubation. By comparison, wild-type GlcCerasetreated under the same experimental conditions retained 68% of itsinitial activity after 30 minutes of incubation. Wild-type GlcCeraseretained 46% of its initial activity after 60 minutes of incubation.Wild-type GlcCerase retained 20% of its initial activity after 120minutes of incubation. Wild-type GlcCerase retained 8% of its initialactivity after 180 minutes of incubation.

The estimated half-life (operationally defined in this study as theincubation time at about pH 7.5 and about 37° C. that resulted in a 50%loss of initial enzyme activity) for GlcCerase-K321V is approximately 70minutes while the estimated half-life for wild-type GlcCerase isapproximately 50 minutes under these experimental conditions.

The above results are summarized in Table 1

TABLE 1 Recombinant GlcCerase Residual Estimated Fold- protein n =Activity (3 hr) half-life improvement Wild-type 7  8%  ~50 min — H145L 442% ~160 min 3.2 H145F 2 17% ~100 min 2.0 F316A/L317F 4 34% ~120 min 2.4K321N 4 54% >180 min >3.6 K321A 2 49% ~170 min 3.4 K321V 1 15%  ~70 min1.4 F316A/L317F/K321N 5 52% >180 min >3.6 H145L/F316A/L317F 1 39% ~135min 2.7 H145L/K321N 2 62% >180 min >3.6

Residual activity is the amount of enzyme activity that remains afterthe three hour incubation and expressed as the percent of initial enzymeactivity (at t=0 min) for each variant, recombinant β-glucocerebrosidaseprotein in the stability assay. Each recombinant β-glucocerebrosidaseprotein was tested in at least two separate experiments (indicated by nin Table 1) to determine the average residual activities during thethree hour time course except for GlcCerase-K321V andGlcCerase-H145L/F316A/L317F which were only tested once. Thefold-improvement refers to the increase in the apparent half-lives ofthe variant, recombinant GlcCerase proteins relative to purifiedwild-type, recombinant GlcCerase protein.

The variant, recombinant β-glucocerebrosidase protein that has an aminoacid variation at position 145, 316, 317, or 321, orGlcCerase-F316A/L317F, GlcCerase-K321N, GlcCerase-K321A,GlcCerase-K321V, GlcCerase-H145L, GlcCerase-H145F, orGlcCerase-F316A/L317F/K321N or GlcCerase-F316A/L317F/K321A orGlcCerase-F316A/L317F/K321V or GlcCerase-H145L/F316A/L317F orGlcCerase-H145L/K321N which can be characterized as retaining morecatalytic activity relative to wild-type, recombinantβ-glucocerebrosidase could be made in yeast cells, plant cells,mammalian cells or transgenic animals using molecular biology andprotein purification techniques known to persons skilled in the art.

A composition comprising the variant, recombinant β-glucocerebrosidaseprotein that has an amino acid variation at position 145, 316, 317, or321, or GlcCerase-F316A/L317F, GlcCerase-K321N, GlcCerase-K321A,GlcCerase-K321V, GlcCerase-H145L, GlcCerase-H145F, orGlcCerase-F316A/L317F/K321N or GlcCerase-F316A/L317F/K321A orGlcCerase-F316A/L317F/K321V or GlcCerase-H145L/F316A/L317F orGlcCerase-H145L/K321N and a pharmaceutically acceptable carrier could bemade using pharmaceutically acceptable carriers known to persons skilledin the art.

A composition comprising the variant, recombinant β-glucocerebrosidaseprotein that has an amino acid variation at position 145, 316, 317, or321, or GlcCerase-F316A/L317F, GlcCerase-K321N, GlcCerase-K321A,GlcCerase-K321V, GlcCerase-H145L, GlcCerase-H145F, orGlcCerase-F316A/L317F/K321N or GlcCerase-F316A/L317F/K321A orGlcCerase-F316A/L317F/K321V or GlcCerase-H145L/F316A/L317F orGlcCerase-H145L/K321N and a pharmaceutically acceptable buffer could bemade using pharmaceutically acceptable buffers known to persons skilledin the art.

Example 12

Since GlcCerase protein is stable at acidic pH and very efficient atclearing the accumulated glucosylceramide substrate within lysosomes,one can develop a more stable GlcCerase ERT which can better withstandthe unfavorable (neutral pH) environment to retain its catalyticactivity so that a larger quantity of active drug is delivered tolysosomes for improved efficacy. The GlcCerase enzyme can be stabilizedat about neutral pH when bound (and inhibited) by the active site enzymeinhibitor isofagomine (IFG). Protein stability for recombinant GlcCeraseat about neutral pH is significantly improved with IFG as evidenced byan increase of up to 15° C. in the thermal melting temperature (Tm). Themechanism of action for IFG-induced GlcCerase stabilization is believedto result from the extensive hydrogen bonding network between IFG andsix GlcCerase active site residues to restrict the unfolding of theactive site and surrounding regions to maintain a more stable GlcCeraseconformation. Maintaining the proper GlcCerase structure at the activesite and surrounding regions helps catalytic activity and overallGlcCerase stability. A number of different modifications to GlcCerasecan help retain catalytic activity and maintain a more stable proteinstructure at about neutral pH. Herein is provided the construction of aseries of different GlcCerase enzymes with specific amino acidsubstitutions at strategic locations within loop structures near theactive site to help form a more-ordered region near the active site thatare less prone to unwinding at about neutral pH. Additionally, hereinare provided modifications to an α-helix near the active site which mayhelp to move this helix and an adjacent loop away from the catalyticsite to maintain an open, active conformation.

An approach was utilized to generate a series of modified GlcCeraseenzymes that were predicted to have superior protein stability thanwild-type GlcCerase. Primarily, two different but complimentarystrategies were used: (1) modification of certain loops and helices nearthe catalytic site to mimic the preferred, active GlcCeraseconformations without actually inhibiting the enzyme; and, (2)modification of a random coil region which may enhance interactionsbetween different residues and secondary structures for improved proteinstability.

In one example, several residues were modified within the loop1 region(positions 311-319) to help form a more-ordered region near the activesite that would be less prone to unwinding at about neutral pH. Thisvariant (GlcCerase-F316A/L317F) with modifications within loop1 wasshown to be significantly more stable than GlcCerase at about pH 7.5 and37° C.

In a second example, an α-helix near the active site (α6) was modifiedthat was intended to stabilize this helix and help pull an adjacent loop(loop1) away from the catalytic site to maintain an open, activeconformation. Thus, modified GlcCerase enzymes were generated thatcontained an amino acid substitution at position 321 to replace acharged lysine residue with an uncharged residue within helix α6 topromote more hydrophobic interactions with adjacent α-helices andβ-structures to stabilize the protein. Protein alignments were analyzedfor GlcCerase enzymes for various species (Table II) and noted that aGlcCerase homolog of B. taurus (bull GlcCerase; accession codeDAA31806.1) contained asparagine at position 321 (³²¹Asn). This suggeststhat lysine at position 321 (³²¹Lys) may be potentially be replaced witha different amino acid residue without abolishing GlcCerase catalyticactivity. GlcCerase-K321N retained approximately 75% and 54% of itsoriginal catalytic activity at about pH 7.5 and 37° C. after 2 and 3hrs, respectively. In contrast, wild-type GlcCerase retained only 21%and 8% of its initial activity under the same conditions. The estimatedhalf-life of GlcCerase-K321N was approximately 3.5-fold longer thanwild-type GlcCerase. Similarly, GlcCerase-K321A retained approximately68% and 49% of its original catalytic activity at about pH 7 and 37° C.after 2 and 3 hrs, respectively. The estimated half-life ofGlcCerase-K321A was approximately 3-fold longer than wild-typeGlcCerase. GlcCerase-K321V retained approximately 29% and 15% of itsoriginal catalytic activity at about pH 7 and 37° C. after 2 and 3 hrs,respectively. The estimated half-life of GlcCerase-K321V wasapproximately 1.4-fold longer than wild-type GlcCerase. These data showthat removing a positively-charged lysine residue within helix α6 madethis region more ordered to limit unwinding of this region to confergreater GlcCerase protein stability.

In a third example, a random coil region was modified between abeta-sheet (β2) and an α-helix (α2) which may facilitate betterinteractions between different residues and secondary structures forimproved stability. Protein alignment analysis of GlcCerase enzymes fromvarious species (Table 2) revealed that position 145 within this randomcoil was divergent among the different species. A B. taurus homolog andS. scrofa (pig) both contain leucine at this position while murineGlcCerase contains serine. Thus, we substituted histidine at position145 (¹⁴⁵His) with either Leu (H145L) or Phe (H145F) to determine whetherthese modifications would improve GlcCerase stability. GlcCerase-H145LGlcCerase was shown to be more stable than wild-type GlcCerase andretained 62% of its initial activity after 2 hrs and 42% after 3 hrswith an estimated half-life of approximately 3-fold longer half-lifethan wild-type GlcCerase (160 min vs. 50 min). Similarly, GlcCeraseH145F retained approximately 37% after 2 hrs of its initial activity and17% after 3 hrs with an approximately 2-fold longer half-life relativeto wild-type GlcCerase. It is not currently known how thesemodifications affected the GlcCerase structure but it is possible thatreplacing a partially charged residue (¹⁴⁵His) with either leucine orphenylalanine hydrophobic residue would make this region more ordered tolimit unwinding of this region. Alternatively, these modifications maycreate a turn and enhance interactions between secondary structures(e.g., beta-sheet β2 and helix α2).

Protein alignment data is summarized in Table 2

TABLE 2  Human ¹⁴⁰DDFQLH NFSLPEEDT ³¹⁵DFLAPAKATLGET GlcCeraseSEQ ID NO: 17 SEQ ID NO: 21 S. scorfa ¹⁴⁰DDFQLL NFSLPEEDV³¹⁵DFLAPAKATLGET GlcCerase SEQ ID NO: 18 SEQ ID NO: 22 B taurus¹⁴⁰DDFQLL NFSLPEEDV ³¹⁵DFLAPA NATLGET GlcCerase SEQ ID NO: 19SEQ ID NO: 23 Murine ¹⁴⁰NDFQLS NFSLPEEDT ³¹⁵DFLAPAKATLGET GlcCeraseSEQ ID NO: 20 SEQ ID NO: 24

Suitable variant, recombinant β-glucocerebrosidase proteins can alsohave one or more replacement amino acids in the loop1 region of theprotein, the one or more replacement amino acids characterized as havinga side-chain conformation that increases order near the active site ofthe protein. These proteins can also have one or more replacement aminoacids in the loop1 region of the protein, the one or more replacementamino acids characterized as having a side-chain conformation thatincreases order near the active site of the protein is characterized asbeing more stable at a range of from about pH3 to about pH8 compared towild-type, recombinant β-glucocerebrosidase protein. These proteins canalso have one or more replacement amino acids in the loop1 region of theprotein, the one or more replacement amino acids characterized as havinga side-chain conformation that increases order near the active site ofthe protein is characterized as being more stable at about neutral pHcompared to wild-type, recombinant β-glucocerebrosidase protein. Theseproteins can also have one or more replacement amino acids in the loop1region of the protein, the one or more replacement amino acids ischaracterized as being less prone to unwinding at a range of from aboutpH3 to about pH8 compared to wild-type, recombinant β-glucocerebrosidaseprotein. These proteins can also have one or more replacement aminoacids in the loop1 region of the protein, the one or more replacementamino acids is characterized as being less prone to unwinding at aboutneutral pH compared to wild-type, recombinant β-glucocerebrosidaseprotein.

Suitable variant, recombinant β-glucocerebrosidase proteins can alsohave one or more replacement amino acids in the α-helix near the activesite (α6) of the protein, the one or more replacement amino acid sidescharacterized as having a side-chain confirmation that stabilizes thishelix and pulls adjacent loop1 away from the catalytic site and has anopen and active conformation. These proteins can also have one or morereplacement amino acids in the α-helix near the active site (α6) of theprotein, the one or more replacement amino acid sides characterized ashaving a side-chain confirmation that stabilizes this helix and pullsadjacent loop1 away from the catalytic site and has an open and activeconformation is characterized as being more stable at a range of fromabout pH3 to about pH8 compared to wild-type, recombinantβ-glucocerebrosidase protein. These proteins can also be characterizedas having a more open conformation at about neutral pH compared towild-type, recombinant β-glucocerebrosidase protein. These proteins canalso have one or more replacement amino acids in the loop1 region of theprotein, the one or more replacement amino acids is characterized ashaving a more open conformation at a range of from about pH3 to aboutpH8 compared to wild-type, recombinant β-glucocerebrosidase protein.These proteins can also have one or more replacement amino acids in theloop1 region of the protein, the one or more replacement amino acidscharacterized as having a more open conformation at about neutral pHcompared to wild-type, recombinant β-glucocerebrosidase protein.

Suitable variant, recombinant β-glucocerebrosidase protein can also haveone or more replacement amino acids in the random coil region betweenbeta-sheet (β2) and an α-helix (α2), the one or more replacement aminoacid side-chains characterized as having a side-chain confirmation thatfacilitates better interactions between different residues and secondarystructures for improved stability. These proteins can also have one ormore replacement amino acids in the random coil region betweenbeta-sheet (β2) and an α-helix (α2), the one or more replacement aminoacid side-chains characterized as having a side-chain confirmation thatfacilitates better interactions between different residues and secondarystructures for improved stability is characterized as being more stableat a range of from about pH3 to about pH8 compared to wild-type,recombinant β-glucocerebrosidase protein. These proteins can also haveone or more replacement amino acids in the random coil region betweenbeta-sheet (β2) and an α-helix (α2), the one or more replacement aminoacid side-chains characterized as having a side-chain confirmation thatfacilitates better interactions between different residues and secondarystructures for improved stability at about neutral pH compared towild-type, recombinant β-glucocerebrosidase protein. These proteins canalso have one or more replacement amino acids in the random coil regionbetween beta-sheet (β2) and an α-helix (α2), the one or more replacementamino acid side-chains characterized as having a side-chain confirmationthat facilitates better interactions between different residues andsecondary structures for improved stability at a range of from about pH3to about pH8 compared to wild-type, recombinant β-glucocerebrosidaseprotein. These proteins can also have one or more replacement aminoacids in the random coil region between beta-sheet (β2) and an α-helix(α2), the one or more replacement amino acid side-chains characterizedas having a side-chain confirmation that facilitates better interactionsbetween different residues and secondary structures for improvedstability at about neutral pH compared to wild-type, recombinantβ-glucocerebrosidase protein.

Suitable methods for treating a lysosomal storage disease can alsoinclude administering to a subject in need thereof a variant,recombinant β-glucocerebrosidase protein characterized as havingincreased stability compared to wild-type, recombinantβ-glucocerebrosidase is provided. Methods for treating a lysosomalstorage disease can also include administering to a subject in needthereof a variant, recombinant β-glucocerebrosidase proteincharacterized as retaining increased catalytic activity compared towild-type, recombinant β-glucocerebrosidase. Methods for treating alysosomal storage disease can also include administering to a subject inneed thereof a variant, recombinant β-glucocerebrosidase proteincharacterized as having an increased specific activity compared towild-type, recombinant β-glucocerebrosidase.

Methods for treating a lysosomal storage disease can also includeadministering to a subject in need thereof a variant, recombinantβ-glucocerebrosidase protein having an amino acid variation at one ormore of the following positions: 316, 317, 321 and 145. Methods fortreating a lysosomal storage disease can also include administering to asubject in need thereof a variant, recombinant β-glucocerebrosidaseprotein with the amino acid variations 316A and L317F. Methods fortreating a lysosomal storage disease can also include administering to asubject in need thereof a variant, recombinant β-glucocerebrosidaseprotein with the amino acid variation K321N. Methods for treating alysosomal storage disease can also include administering to a subject inneed thereof a variant, recombinant β-glucocerebrosidase protein withthe amino acid variation K321A. Methods for treating a lysosomal storagedisease can also include administering to a subject in need thereof avariant, recombinant β-glucocerebrosidase protein with the amino acidvariation K321V. Methods for treating a lysosomal storage disease canalso include administering to a subject in need thereof a variant,recombinant β-glucocerebrosidase protein with the amino acid variationH145L. Methods for treating a lysosomal storage disease can also includeadministering to a subject in need thereof a variant, recombinantβ-glucocerebrosidase protein with the amino acid variation H145F.Methods for treating a lysosomal storage disease can also includeadministering to a subject in need thereof a variant, recombinantβ-glucocerebrosidase protein with the amino acid variations F316A,L317F, and K321N. Methods for treating a lysosomal storage disease canalso include administering to a subject in need thereof a variant,recombinant β-glucocerebrosidase protein with the amino acid variationsF316A, L317F, and K321A. Methods for treating a lysosomal storagedisease can also include administering to a subject in need thereof avariant, recombinant β-glucocerebrosidase protein with the amino acidvariations F316A, L317F, and K321V. Methods for treating a lysosomalstorage disease can also include administering to a subject in needthereof a variant, recombinant β-glucocerebrosidase protein with theamino acid variations H145L, F316A, and L317F. Methods for treating alysosomal storage disease can also include administering to a subject inneed thereof a variant, recombinant β-glucocerebrosidase protein withthe amino acid variations H145L and K321N. Methods for treating alysosomal storage disease can also include administering to a subject inneed thereof a variant, recombinant β-glucocerebrosidase protein withthe amino acid variations H145L and K321A. Methods for treating alysosomal storage disease can also include administering to a subject inneed thereof a variant, recombinant β-glucocerebrosidase protein withthe amino acid variations H145L and K321V. Methods to treat thelysosomal storage disease can also include methods to treat Gaucherdisease. Methods of treatment can be by intravenous infusion, byintramuscular injection or by other routes of administration known toone skilled in the art.

Suitable compounds can have a variant, recombinant β-glucocerebrosidaseprotein characterized as having any one of the following amino acidsequences: SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15,or SEQ ID NO: 16. Suitable compounds can have a variant, recombinantβ-glucocerebrosidase protein characterized as having any one of thefollowing amino acid sequences: SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO:4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9,SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO:14, SEQ ID NO: 15, or SEQ ID NO: 16.

Suitable variant, recombinant β-glucocerebrosidase proteins can also becharacterized as capable of increased expression relative to wild-type,recombinant β-glucocerebrosidase. Variant, recombinantβ-glucocerebrosidase protein expression can also be increased by atleast about 10% relative to wild-type, recombinant β-glucocerebrosidase.Variant, recombinant β-glucocerebrosidase protein expression can also beincreased by at least about 25% relative to wild-type, recombinantβ-glucocerebrosidase. Variant, recombinant β-glucocerebrosidase proteinexpression can also be increased by at least about 50% relative towild-type, recombinant β-glucocerebrosidase. Variant, recombinantβ-glucocerebrosidase protein expression can also be increased by atleast about 80% relative to wild-type, recombinant β-glucocerebrosidase.Variant, recombinant β-glucocerebrosidase proteins can also be capableof being expressed in mammalian cells, transgenic animals or in yeastcells. Variant, recombinant β-glucocerebrosidase protein can also beexpressed in a human. Variant, recombinant β-glucocerebrosidase proteincan also be expressed from an inserted gene.

Example 13

Variant, recombinant GlcCerase proteins and wild-type GlcCerase wereexpressed in cell culture according to methods mentioned previously toassess the relative expression of these variant GlcCerase enzymescompared to the wild-type GlcCerase. A number of different variantGlcCerase enzymes were expressed better than wild-type GlcCerase intransient transfection experiments as measured by enzyme activity fromconditioned medium after about 48 hours post transfection (FIG. 1).GlcCerase-F316A/L317A was expressed better than wild-type GlcCerase(about 282%), GlcCerase-K321N was expressed better than wild-typeGlcCerase (about 272%), GlcCerase-H145L was expressed better thanwild-type GlcCerase (about 157%), GlcCerase-F316A/L317A/K321N wasexpressed better than wild-type GlcCerase (about 272%),GlcCerase-H145L/K321N was expressed better than wild-type GlcCerase(about 317%). GlcCerase-K321A was expressed equivalently as wild-typeGlcCerase while GlcCerase-K321V was expressed at lower levels thanwild-type (about 61%) (data not shown).

Also provided herein are methods of making a compound comprising avariant, recombinant β-glucocerebrosidase protein characterized ashaving any one of the following amino acid sequences: SEQ ID NO: 2, SEQID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ IDNO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ IDNO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16. Also providedherein are methods of making a nucleic acid encoding a variant,recombinant β-glucocerebrosidase protein characterized as having any oneof the following amino acid sequences: SEQ ID NO: 2, SEQ ID NO: 3, SEQID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ IDNO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16.

Example 14 Plasmid Construction for Wild-Type and Modified GlcCeraseEnzymes

DNA plasmids for the expression of wild-type and modified GlcCeraseenzymes were generated using oligonucleotide primers (listed in Table 3)or synthetic DNA minigenes (>400 bp) encoding a fragment of GlcCerasewith specific amino acid substitutions. All primers and synthetic DNAminigenes were purchased from Integrated DNA Technologies™ (Coralville,Iowa). Wild-type human GlcCerase (designated as pHD101) was constructedby generating GlcCerase cDNA by PCR and ligating into a mammalianexpression vector. Briefly, the entire wild-type human GlcCerase cDNAwas amplified with its natural Kozak sequence and stop codon usingprimers A & B and the human GlcCerase cDNA clone (Origene™) template DNAin two identical 50 μl reactions via Phusion-HF high-fidelity DNAPolymerase™ (NEB™) and 200 μM dNTPs. Primer A was constructed to containa 5′ BglII and an internal EcoRI restriction site that immediatelypreceded the Kozak sequence while primer B contained 3′ NheI and NotIrestriction sites that followed the stop codon to enable cloning of thePCR product into expression vectors. The PCR reactions were pooled andthe resultant ˜1.6 kilobase (kb) PCR product was separated and excisedfrom a 1% (w/v) agarose preparative gel and isolated using QIAGEN's™ GelExtraction kit. The PCR product was subsequently digested overnight withrestriction endonucleases BglII and NotI at 37° C. and re-purified usingQIAGEN's™ PCR cleanup kit per the manufacturer's instructions. Themammalian expression vector pEF6/V5-HisA™ was digested with BamHI andNotI, dephosphorylated using Antarctic Phosphatase™ and isolated usingQIAGEN's™ PCR cleanup kit. The BglII-NotI digested PCR product (2 μl)was ligated into the BamHI-NotI pEF6/V5-HisA™ vector (1 μl) using T4 DNAligase (NEB™) according to the manufacturer's instructions.Chemically-competent E. coli cells were transformed with 1 μl of theligation reaction and plated onto Luria-Bertani (LB) agar platescontaining 100 μg/ml ampicillin and incubated overnight at 37° C. toform distinct bacterial colonies that had been transformed with theplasmid DNA containing the β-lactamase gene to confer ampicillinresistance. Individual ampicillin-resistant bacterial colonies werepicked and expanded in 4 ml LB broth (containing 100 μg/ml ampicillin)overnight at 37° C. and plasmid DNA was isolated on the following day byMiniprep™ (QIAGEN™) according to the manufacturer's instructions. Theisolated plasmid DNAs were checked by two different restriction digestreactions using EcoRI & NheI and BamHI, respectively. A correct plasmidDNA from clone 4 (designated as pHD 101.4) was chosen and used tore-transform competent E. coli cells as described above for high-levelreplication of the plasmid DNA. A colony from a LB agar-ampicillin platewas then picked and grown in 200 ml LB broth overnight at 37° C. andplasmid pHD101.4 was isolated by Maxiprep™ (Promega™). Plasmid pHD 101.4(encoding wild-type human GlcCerase cDNA) was verified by DNA sequencingand used for the construction of other GlcCerase enzymes and fortransient transfection experiments.

A BglII restriction site was incorporated into primer 1 so that ligationof the BglII-digested GlcCerase PCR product into the compatible BamHIsite of the pEF6/V5-HisA™ vector eliminated the BamHI restriction sitewithin the multiple cloning site and this modified expression vectorwill be referred to as pEF6′ hereafter. The elimination of this BamHIsite within pEF6/V5-HisA™ was necessary so that a unique BamHI sitewithin the GlcCerase cDNA can be utilized for inserting DNA fragmentswith specific nucleotide substitutions to generate modified GlcCeraseenzymes.

To generate GlcCerase-F316A/L317F (designated as pHD105), a GlcCeraseminigene containing these amino acid substitutions was synthesized byIntegrated DNA Technologies™ between natural flanking 5′ BsrGI and 3′BamHI restriction sites. The synthetic modified GlcCerase DNA fragment(˜0.5 kb) was released from the pIDTSMART™ plasmid by BsrGI and BamHIrestriction digest and subsequently isolated by preparative 1% agarosegel and ligated in-frame into pHD 101.4 that had been previouslydigested with BsrGI and BamHI and dephosphorylated. One microliter ofthis ligation reaction was used to transform competent E. coli cells andthe sample was processed as described above for pHD101. Miniprep DNA wasisolated from individual clones and tested by restriction digest withEcoRI and XbaI. Clone 5 (designated as pHD105.5) was chosen, verified byDNA sequencing and used for further characterization.

To generate GlcCerase-K321N (designated as pHD109), the amino acidsubstitution was introduced by overlap PCR. Briefly, the N-terminalfragment (˜1.1 kb) was generated by using primers A & D and pHD101.4 asthe template DNA in PCR reaction 1 while the C-terminal fragment (˜0.5kb) was generated using primers B & C and pHD101.4 template in PCRreaction 2. The entire K321N GlcCerase cDNA was then synthesized in PCRreaction 3 by adding 1 μl from PCR reactions A & B and primers 1 & 2.The resultant PCR product 3 (˜1.6 kb) was isolated from a preparative 1%agarose gel as before and digested with EcoRI and NotI restrictionendonucleases. PCR product 3 was re-purified and ligated intoEcoRI/NotI-digested and dephosphorylated pEF6′ vector and processed asdescribed above. Miniprep DNA was isolated from individual clones andtested by restriction digest with EcoRI and XbaI. Clone 3 (designated aspHD109.3) was chosen, verified by DNA sequencing and used for furthercharacterization.

To generateGlcCerase-H145L (designated as pHD110), the amino acidsubstitution was introduced by overlap PCR. The N-terminal fragment(˜0.55 kb) was generated by using primers A & F and pHD 101.4 templateDNA in PCR reaction 4 while the C-terminal fragment (˜1 kb) wasgenerated using primers B & E and pHD 101.4 template in PCR reaction 5.The entire H145L GlcCerase cDNA was generated in PCR reaction 6 byadding 1 μl from PCR reactions 4 & 5 and primers A & B. The resultantPCR product 6 (˜1.6 kb) was isolated from a preparative 1% agarose gelas before and digested with EcoRI and NotI restriction endonucleases.PCR product 6 was re-purified and ligated into EcoRI/NotI-digested anddephosphorylated pEF6′ vector and processed as before. Miniprep DNA wasisolated from individual clones and tested by restriction digest withEcoRI and XbaI. Clone 2 (designated as pHD110.2) was chosen, verified byDNA sequencing and used for further characterization.

To generate GlcCerase-H145F (designated as pHD111), the amino acidsubstitution was introduced by overlap PCR. The N-terminal fragment(˜0.55 kb) was generated by using primers A & H and pHD 101.4 templateDNA in PCR reaction 7 while the C-terminal fragment (˜1 kb) wasgenerated using primers B & G and pHD 101.4 template in PCR reaction 8.The entire H145L GlcCerase cDNA was generated in PCR reaction 9 byadding 1 μl from PCR reactions 7 & 8 and primers A & B. The resultantPCR product 9 (˜1.6 kb) was isolated from a preparative 1% agarose gelas before and digested with EcoRI and NotI restriction endonucleases.PCR product 9 was re-purified and ligated into EcoRI/NotI-digested anddephosphorylated pEF6′ vector and processed as before. Miniprep DNA wasisolated from individual clones and tested by restriction digest withEcoRI and XbaI. Clone 1 (designated as pHD111.1) was chosen, verified byDNA sequencing and used for further characterization.

To generate GlcCerase-F316A/L317F/K321N (designated as pHD112), theamino acid substitution was introduced into pHD105.5 by overlap PCR. TheN-terminal fragment (˜1.1 kb) was generated by using primers A & D andpHD 105.5 template DNA in PCR reaction 10 while the C-terminal fragment(˜0.5 kb) was generated using primers B & C and pHD 105.5 template inPCR reaction 11. The entire F316A/L317F/K321N GlcCerase cDNA wasgenerated in PCR reaction 12 by adding 1 μl from PCR reactions 10 & 11and primers A & B. The resultant PCR product 12 (˜1.6 kb) was isolatedfrom a preparative 1% agarose gel as before and digested with EcoRI andNotI restriction endonucleases. PCR product 12 was re-purified andligated into EcoRI/NotI-digested and dephosphorylated pEF6′ vector asdescribed above. Miniprep DNA was isolated from individual clones andtested by restriction digest using EcoRI and XbaI. Clone 7 (designatedas pHD112.7) was chosen, verified by DNA sequencing and used for furthercharacterization.

To generate GlcCerase-H145L/F316A/L317F (designated as pHD113), theamino acid substitution was introduced into pHD105.5 by overlap PCR. TheN-terminal fragment (˜0.55 kb) was generated by using primers A & F andpHD105.5 template DNA in PCR reaction 13 while the C-terminal fragment(˜1 kb) was generated using primers B & E and pHD105.5 template in PCRreaction 14. The entire H145L/F316A/L317F GlcCerase cDNA was generatedin PCR reaction 15 by adding 1 μl from PCR reactions 13 & 14 and primersA & B. The resultant PCR product 15 (˜1.6 kb) was isolated from apreparative 1% agarose gel as before and digested with EcoRI and NotIrestriction endonucleases. PCR product 15 was re-purified and ligatedinto EcoRI/NotI-digested and dephosphorylated pEF6′ vector as previouslydescribed.

To generate GlcCerase-H145F/F316A/L317F (designated as pHD114), theamino acid substitution was introduced into pHD105.5 by overlap PCR. TheN-terminal fragment (˜0.55 kb) was generated by using primers A & H andpHD105.5 template DNA in PCR reaction 16 while the C-terminal fragment(˜1 kb) was generated using primers B & G and pHD105.5 template in PCRreaction 17. The entire H145F/F316A/L317F GlcCerase cDNA was generatedin PCR reaction 18 by adding 1 μl from PCR reactions 16 & 17 and primersA & B. The resultant PCR product 18 (˜1.6 kb) was isolated from apreparative 1% agarose gel as before and digested with EcoRI and NotIrestriction endonucleases. PCR product 18 was re-purified and ligatedinto EcoRI/NotI-digested and dephosphorylated pEF6′ vector as previouslydescribed.

To generate GlcCerase-H145L/K321N (designated as pHD115), both pHD109.3and pHD110.2 were digested with BsrGI and BamHI restriction enzymes andthe ˜0.5 kb fragment from pHD109.3 and the ˜6.9 kb fragment frompHD110.2 (after treatment with Antarctic phosphatase) were isolated bypreparative 1% agarose gel. The ˜0.5 kb fragment from pHD109.3(containing the K321N amino acid substitution) was then ligated in-frameinto plasmid pHD110.2 (contains the H145L modification) and processed asbefore. Miniprep DNA was isolated from individual clones and tested byrestriction digest with EcoRI and XbaI. Clone 5 was chosen, verified byDNA sequencing and used for further characterization.

To generate GlcCerase-H145L/F316A/L317F/K321N (designated as pHD116),both pHD112.7 and pHD110.2 will be digested with BsrGI and BamHIrestriction enzymes and the ˜0.5 kb fragment from pHD 112.7 and the ˜6.9kb fragment from pHD 110.2 (after treatment with Antarctic Phosphatase™)were isolated by preparative 1% agarose gel. The ˜0.5 kb fragment frompHD112.7 (containing the F316A/L317F/K321N amino acid substitutions)will be ligated in-frame into plasmid pHD110.2 (contains the H145Lmodification) and processed as before. Miniprep DNA will be isolatedfrom individual clones and tested by restriction digest with EcoRI andXbaI to identify transformed bacterial strain with the correctGlcCerase-H145L/F316A/L317F/K321A cDNA. The selected DNA construct willbe verified by DNA sequencing and will be used for furthercharacterization.

To generate GlcCerase-K321A (designated as pHD117), the amino acidsubstitution was introduced by overlap PCR. Briefly, the N-terminalfragment (˜1.1 kb) was generated by using primers A & J and pHD 101.4 asthe template DNA in PCR reaction 19 while the C-terminal fragment (˜0.5kb) was generated using primers B & I and pHD101.4 template in PCRreaction 20. PCR products 19 and 20 were isolated by preparative 1%agarose gel and used to as template DNA to synthesize the entire K321AGlcCerase cDNA fragment in PCR reaction 21 using primers A & B. Theresultant PCR product 21 (˜1.6 kb) was isolated from a preparative 1%agarose gel as before and digested with EcoRI and NotI restrictionendonucleases. PCR product 21 was re-purified and ligated intoEcoRI/NotI-digested and dephosphorylated pEF6′ vector and processed asdescribed above. Miniprep DNA was isolated from individual clones andtested by restriction digest with EcoRI and XbaI. Clone 1 (designated aspHD117.1) was chosen and used for further characterization.

To generate GlcCerase-K321V (designated as pHD118), the amino acidsubstitution was introduced by overlap PCR. Briefly, the N-terminalfragment (˜1.1 kb) was generated by using primers A & L and pHD 101.4 asthe template DNA in PCR reaction 22 while the C-terminal fragment (˜0.5kb) was generated using primers B & K and pHD101.4 template in PCRreaction 23. PCR products 22 and 23 were isolated by preparative 1%agarose gel and used to as template DNA to synthesize the entire K321AGlcCerase cDNA fragment in PCR reaction 24 using primers A & B. Theresultant PCR product 24 (˜1.6 kb) was isolated from a preparative 1%agarose gel as before and digested with EcoRI and NotI restrictionendonucleases. PCR product 24 was re-purified and ligated intoEcoRI/NotI-digested and dephosphorylated pEF6′ vector and processed asdescribed above. Miniprep DNA was isolated from individual clones andtested by restriction digest with EcoRI and XbaI. Clone 7 (designated aspHD118.7) was chosen and used for further characterization.

To generate GlcCerase-F316A/L317F/K321A (designated as pHD119), theamino acid substitution will be introduced into pHD105.5 by overlap PCR.The N-terminal fragment (˜1.1 kb) will be generated by using primers A &J and pHD 105.5 template DNA in PCR reaction 25 while the C-terminalfragment (˜0.5 kb) will be generated using primers B & I and pHD105.5template in PCR reaction 26. PCR products 25 and 26 will be isolated bypreparative 1% agarose gel and will beused to as template DNA tosynthesize the entire F316A/L317F/K321A/K321A GlcCerase cDNA fragment inPCR reaction 27 using primers A & B. The resultant PCR product 27 (˜1.6kb) will be isolated from a preparative 1% agarose gel as before anddigested with EcoRI and NotI restriction endonucleases. PCR product 27will be re-purified and ligated into EcoRI/NotI-digested anddephosphorylated pEF6′ vector as described above. Miniprep DNA will beisolated from individual clones and tested by restriction digest usingEcoRI and XbaI to identify transformed bacterial strain with the correctGlcCerase-F316A/L317F/K321A cDNA. The selected DNA construct will beverified by DNA sequencing and will be used for furthercharacterization.

To generate GlcCerase-F316A/L317F/K321V (designated as pHD120), theamino acid substitution will be introduced into pHD105.5 by overlap PCR.The N-terminal fragment (˜1.1 kb) will be generated by using primers A &L and pHD 105.5 template DNA in PCR reaction 28 while the C-terminalfragment (˜0.5 kb) will be generated using primers B & K and pHD 105.5template in PCR reaction 29. PCR products 28 and 29 will be isolated bypreparative 1% agarose gel and will be used to as template DNA tosynthesize the entire F316A/L317F/K321A/K321V GlcCerase cDNA fragment inPCR reaction 30 using primers A & B. The resultant PCR product 30 (˜1.6kb) will be isolated from a preparative 1% agarose gel as before anddigested with EcoRI and NotI restriction endonucleases. PCR product 30will be re-purified and ligated into EcoRI/NotI-digested anddephosphorylated pEF6′ vector as described above. Miniprep DNA will beisolated from individual clones and tested by restriction digest usingEcoRI and XbaI to identify transformed bacterial strain with the correctGlcCerase-F316A/L317F/K321V cDNA. The selected DNA construct will beverified by DNA sequencing and will be used for furthercharacterization.

To generate GlcCerase-H145L/K321A (designated as pHD121), both pHD117.1and pHD110.2 will be digested with BsrGI and BamHI restriction enzymesand the ˜0.5 kb fragment from pHD117.1 and the ˜6.9 kb fragment frompHD110.2 (after treatment with Antarctic Phosphatase™) will be isolatedby preparative 1% agarose gel. The ˜0.5 kb fragment from pHD117.1(containing the K321A amino acid substitution) will be ligated in-frameinto plasmid pHD110.2 (contains the H145L modification) and processed asbefore. Miniprep DNA was isolated from individual clones and tested byrestriction digest with EcoRI and XbaI to identify transformed bacterialstrain with the correct GlcCerase-H145L/K321A cDNA. The selected DNAconstruct will be verified by DNA sequencing and will be used forfurther characterization.

To generate GlcCerase-H145L/K321V (designated as pHD122), both pHD118.7and pHD110.2 will be digested with BsrGI and BamHI restriction enzymesand the ˜0.5 kb fragment from pHD118.7 and the ˜6.9 kb fragment frompHD110.2 (after treatment with Antarctic phosphatase) will be isolatedby preparative 1% agarose gel. The ˜0.5 kb fragment from pHD 118.7(containing the K321V amino acid substitution) will be ligated in-frameinto plasmid pHD110.2 (contains the H145L modification) and processed asbefore. Miniprep DNA was isolated from individual clones and tested byrestriction digest with EcoRI and XbaI to identify transformed bacterialstrain with the correct GlcCerase-H145L/K321V cDNA. The selected DNAconstruct will be verified by DNA sequencing and will be used forfurther characterization.

Table 3 summarizes the primer sequences mentioned above for constructingmodified GlcCerase enzymes.

TABLE 3  SEQ ID NO: Primer Strand Oligonucleotide Sequence (5′->3′) 25A + ggcaagatctgaattcgggatggagttttcaagtccttccagag 26 B −tcgagcggccgcaagctagcttatcactggcgacgccacaggtag 27 C +tccagccaacgccaccctag 28 D − ctagggtggcgttggctgga 29 E +tgatttccagttgttgaacttcagcctc 30 F − gaggctgaagttcaacaactggaaatca 31 G +tgatttccagttgttcaacttcagcctc 32 H − gaggctgaagttgaacaactggaaatca 33 I +tccagccgcagccaccctag 34 J − ctagggtggctgcggctgga 35 K +tccagccgtagccaccctagg 36 L − cctagggtggctacggctgga

Although the present invention has been described in relation toparticular embodiments thereof, many other variations and modificationsand other uses is apparent to those skilled in the art. It is preferred,therefore, that the present invention be delineated not by the specificdisclosure herein, but only by the appended claims.

SEQUENCES SEQ ID NO: 1ARPCIPKSFGYSSVVCVCNATYCDSFDPPTFPALGTFSRYESTRSGRRMELSMGPIQANHTGTGLLLTLQPEQKFQKVKGFGGAMTDAAALNILALSPPAQNLLLKSYFSEEGIGYNIIRVPMASCDFSIRTYTYADTPDDFQLHNFSLPEEDTKLKIPLIHRALQLAQRPVSLLASPWTSPTWLKTNGAVNGKGSLKGQPGDIYHQTWARYFVKFLDAYAEHKLQFWAVTAENEPSAGLLSGYPFQCLGFTPEHQRDFIARDLGPTLANSTHHNVRLLMLDDQRLLLPHWAKVVLTDPEAAKYVHGIAVHWYLDFLAPAKATLGETHRLFPNTMLFASEACVGSKFWEQSVRLGSWDRGMQYSHSIITNLLYHVVGWTDWNLALNPEGGPNWVRNFVDSPIIVDITKDTFYKQPMFYHLGHFSKFIPEGSQRVGLVASQKNDLDAVALMHPDGSAVVVVLNRSSKDVPLTIKDPAVGFLETISPGYSI HTYLWRRQSEQ ID NO: 2 ARPCIPKSFGYSSVVCVCNATYCDSFDPPTFPALGTFSRYESTRSGRRMELSMGPIQANHTGTGLLLTLQPEQKFQKVKGFGGAMTDAAALNILALSPPAQNLLLKSYFSEEGIGYNIIRVPMASCDFSIRTYTYADTPDDFQLHNFSLPEEDTKLKIPLIHRALQLAQRPVSLLASPWTSPTWLKTNGAVNGKGSLKGQPGDIYHQTWARYFVKFLDAYAEHKLQFWAVTAENEPSAGLLSGYPFQCLGFTPEHQRDFIARDLGPTLANSTHHNVRLLMLDDQRLLLPHWAKVVLTDPEAAKYVHGIAVHWYLDAFAPAKATLGETHRLFPNTMLFASEACVGSKFWEQSVRLGSWDRGMQYSHSIITNLLYHVVGWTDWNLALNPEGGPNWVRNFVDSPIIVDITKDTFYKQPMFYHLGHFSKFIPEGSQRVGLVASQKNDLDAVALMHPDGSAVVVVLNRSSKDVPLTIKDPAVGFLETISPGYSI HTYLWRRQSEQ ID NO: 3 ARPCIPKSFGYSSVVCVCNATYCDSFDPPTFPALGTFSRYESTRSGRRMELSMGPIQANHTGTGLLLTLQPEQKFQKVKGFGGAMTDAAALNILALSPPAQNLLLKSYFSEEGIGYNIIRVPMASCDFSIRTYTYADTPDDFQLHNFSLPEEDTKLKIPLIHRALQLAQRPVSLLASPWTSPTWLKTNGAVNGKGSLKGQPGDIYHQTWARYFVKFLDAYAEHKLQFWAVTAENEPSAGLLSGYPFQCLGFTPEHQRDFIARDLGPTLANSTHHNVRLLMLDDQRLLLPHWAKVVLTDPEAAKYVHGIAVHWYLDFLAPANATLGETHRLFPNTMLFASEACVGSKFWEQSVRLGSWDRGMQYSHSIITNLLYHVVGWTDWNLALNPEGGPNWVRNFVDSPIIVDITKDTFYKQPMFYHLGHFSKFIPEGSQRVGLVASQKNDLDAVALMHPDGSAVVVVLNRSSKDVPLTIKDPAVGFLETISPGYSI HTYLWRRQSEQ ID NO: 4 ARPCIPKSFGYSSVVCVCNATYCDSFDPPTFPALGTFSRYESTRSGRRMELSMGPIQANHTGTGLLLTLQPEQKFQKVKGFGGAMTDAAALNILALSPPAQNLLLKSYFSEEGIGYNIIRVPMASCDFSIRTYTYADTPDDFQLLNFSLPEEDTKLKIPLIHRALQLAQRPVSLLASPWTSPTWLKTNGAVNGKGSLKGQPGDIYHQTWARYFVKFLDAYAEHKLQFWAVTAENEPSAGLLSGYPFQCLGFTPEHQRDFIARDLGPTLANSTHHNVRLLMLDDQRLLLPHWAKVVLTDPEAAKYVHGIAVHWYLDFLAPAKATLGETHRLFPNTMLFASEACVGSKFWEQSVRLGSWDRGMQYSHSIITNLLYHVVGWTDWNLALNPEGGPNWVRNFVDSPIIVDITKDTFYKQPMFYHLGHFSKFIPEGSQRVGLVASQKNDLDAVALMHPDGSAVVVVLNRSSKDVPLTIKDPAVGFLETISPGYSI HTYLWRRQSEQ ID NO: 5 ARPCIPKSFGYSSVVCVCNATYCDSFDPPTFPALGTFSRYESTRSGRRMELSMGPIQANHTGTGLLLTLQPEQKFQKVKGFGGAMTDAAALNILALSPPAQNLLLKSYFSEEGIGYNIIRVPMASCDFSIRTYTYADTPDDFQLFNFSLPEEDTKLKIPLIHRALQLAQRPVSLLASPWTSPTWLKTNGAVNGKGSLKGQPGDIYHQTWARYFVKFLDAYAEHKLQFWAVTAENEPSAGLLSGYPFQCLGFTPEHQRDFIARDLGPTLANSTHHNVRLLMLDDQRLLLPHWAKVVLTDPEAAKYVHGIAVHWYLDFLAPAKATLGETHRLFPNTMLFASEACVGSKFWEQSVRLGSWDRGMQYSHSIITNLLYHVVGWTDWNLALNPEGGPNWVRNFVDSPIIVDITKDTFYKQPMFYHLGHFSKFIPEGSQRVGLVASQKNDLDAVALMHPDGSAVVVVLNRSSKDVPLTIKDPAVGFLETISPGYSI HTYLWRRQSEQ ID NO: 6 ARPCIPKSFGYSSVVCVCNATYCDSFDPPTFPALGTFSRYESTRSGRRMELSMGPIQANHTGTGLLLTLQPEQKFQKVKGFGGAMTDAAALNILALSPPAQNLLLKSYFSEEGIGYNIIRVPMASCDFSIRTYTYADTPDDFQLHNFSLPEEDTKLKIPLIHRALQLAQRPVSLLASPWTSPTWLKTNGAVNGKGSLKGQPGDIYHQTWARYFVKFLDAYAEHKLQFWAVTAENEPSAGLLSGYPFQCLGFTPEHQRDFIARDLGPTLANSTHHNVRLLMLDDQRLLLPHWAKVVLTDPEAAKYVHGIAVHWYLDAFAPANATLGETHRLFPNTMLFASEACVGSKFWEQSVRLGSWDRGMQYSHSIITNLLYHVVGWTDWNLALNPEGGPNWVRNFVDSPIIVDITKDTFYKQPMFYHLGHFSKFIPEGSQRVGLVASQKNDLDAVALMHPDGSAVVVVLNRSSKDVPLTIKDPAVGFLETISPGYSI HTYLWRRQSEQ ID NO: 7 ARPCIPKSFGYSSVVCVCNATYCDSFDPPTFPALGTFSRYESTRSGRRMELSMGPIQANHTGTGLLLTLQPEQKFQKVKGFGGAMTDAAALNILALSPPAQNLLLKSYFSEEGIGYNIIRVPMASCDFSIRTYTYADTPDDFQLLNFSLPEEDTKLKIPLIHRALQLAQRPVSLLASPWTSPTWLKTNGAVNGKGSLKGQPGDIYHQTWARYFVKFLDAYAEHKLQFWAVTAENEPSAGLLSGYPFQCLGFTPEHQRDFIARDLGPTLANSTHHNVRLLMLDDQRLLLPHWAKVVLTDPEAAKYVHGIAVHWYLDAFAPAKATLGETHRLFPNTMLFASEACVGSKFWEQSVRLGSWDRGMQYSHSIITNLLYHVVGWTDWNLALNPEGGPNWVRNFVDSPIIVDITKDTFYKQPMFYHLGHFSKFIPEGSQRVGLVASQKNDLDAVALMHPDGSAVVVVLNRSSKDVPLTIKDPAVGFLETISPGYSI HTYLWRRQSEQ ID NO: 8 ARPCIPKSFGYSSVVCVCNATYCDSFDPPTFPALGTFSRYESTRSGRRMELSMGPIQANHTGTGLLLTLQPEQKFQKVKGFGGAMTDAAALNILALSPPAQNLLLKSYFSEEGIGYNIIRVPMASCDFSIRTYTYADTPDDFQLFNFSLPEEDTKLKIPLIHRALQLAQRPVSLLASPWTSPTWLKTNGAVNGKGSLKGQPGDIYHQTWARYFVKFLDAYAEHKLQFWAVTAENEPSAGLLSGYPFQCLGFTPEHQRDFIARDLGPTLANSTHHNVRLLMLDDQRLLLPHWAKVVLTDPEAAKYVHGIAVHWYLDAFAPAKATLGETHRLFPNTMLFASEACVGSKFWEQSVRLGSWDRGMQYSHSIITNLLYHVVGWTDWNLALNPEGGPNWVRNFVDSPIIVDITKDTFYKQPMFYHLGHFSKFIPEGSQRVGLVASQKNDLDAVALMHPDGSAVVVVLNRSSKDVPLTIKDPAVGFLETISPGYSI HTYLWRRQSEQ ID NO: 9 ARPCIPKSFGYSSVVCVCNATYCDSFDPPTFPALGTFSRYESTRSGRRMELSMGPIQANHTGTGLLLTLQPEQKFQKVKGFGGAMTDAAALNILALSPPAQNLLLKSYFSEEGIGYNIIRVPMASCDFSIRTYTYADTPDDFQLLNFSLPEEDTKLKIPLIHRALQLAQRPVSLLASPWTSPTWLKTNGAVNGKGSLKGQPGDIYHQTWARYFVKFLDAYAEHKLQFWAVTAENEPSAGLLSGYPFQCLGFTPEHQRDFIARDLGPTLANSTHHNVRLLMLDDQRLLLPHWAKVVLTDPEAAKYVHGIAVHWYLDFLAPANATLGETHRLFPNTMLFASEACVGSKFWEQSVRLGSWDRGMQYSHSIITNLLYHVVGWTDWNLALNPEGGPNWVRNFVDSPIIVDITKDTFYKQPMFYHLGHFSKFIPEGSQRVGLVASQKNDLDAVALMHPDGSAVVVVLNRSSKDVPLTIKDPAVGFLETISPGYSI HTYLWRRQSEQ ID NO:10 ARPCIPKSFGYSSVVCVCNATYCDSFDPPTFPALGTFSRYESTRSGRRMELSMGPIQANHTGTGLLLTLQPEQKFQKVKGFGGAMTDAAALNILALSPPAQNLLLKSYFSEEGIGYNIIRVPMASCDFSIRTYTYADTPDDFQLLNFSLPEEDTKLKIPLIHRALQLAQRPVSLLASPWTSPTWLKTNGAVNGKGSLKGQPGDIYHQTWARYFVKFLDAYAEHKLQFWAVTAENEPSAGLLSGYPFQCLGFTPEHQRDFIARDLGPTLANSTHHNVRLLMLDDQRLLLPHWAKVVLTDPEAAKYVHGIAVHWYLDAFAPANATLGETHRLFPNTMLFASEACVGSKFWEQSVRLGSWDRGMQYSHSIITNLLYHVVGWTDWNLALNPEGGPNWVRNFVDSPIIVDITKDTFYKQPMFYHLGHFSKFIPEGSQRVGLVASQKNDLDAVALMHPDGSAVVVVLNRSSKDVPLTIKDPAVGFLETISPGYSI HTYLWRRQSEQ ID NO: 11 ARPCIPKSFGYSSVVCVCNATYCDSFDPPTFPALGTFSRYESTRSGRRMELSMGPIQANHTGTGLLLTLQPEQKFQKVKGFGGAMTDAAALNILALSPPAQNLLLKSYFSEEGIGYNIIRVPMASCDFSIRTYTYADTPDDFQLHNFSLPEEDTKLKIPLIHRALQLAQRPVSLLASPWTSPTWLKTNGAVNGKGSLKGQPGDIYHQTWARYFVKFLDAYAEHKLQFWAVTAENEPSAGLLSGYPFQCLGFTPEHQRDFIARDLGPTLANSTHHNVRLLMLDDQRLLLPHWAKVVLTDPEAAKYVHGIAVHWYLDFLAPAAATLGETHRLFPNTMLFASEACVGSKFWEQSVRLGSWDRGMQYSHSIITNLLYHVVGWTDWNLALNPEGGPNWVRNFVDSPIIVDITKDTFYKQPMFYHLGHFSKFIPEGSQRVGLVASQKNDLDAVALMHPDGSAVVVVLNRSSKDVPLTIKDPAVGFLETISPGYSI HTYLWRRQSEQ ID NO: 12 ARPCIPKSFGYSSVVCVCNATYCDSFDPPTFPALGTFSRYESTRSGRRMELSMGPIQANHTGTGLLLTLQPEQKFQKVKGFGGAMTDAAALNILALSPPAQNLLLKSYFSEEGIGYNIIRVPMASCDFSIRTYTYADTPDDFQLHNFSLPEEDTKLKIPLIHRALQLAQRPVSLLASPWTSPTWLKTNGAVNGKGSLKGQPGDIYHQTWARYFVKFLDAYAEHKLQFWAVTAENEPSAGLLSGYPFQCLGFTPEHQRDFIARDLGPTLANSTHHNVRLLMLDDQRLLLPHWAKVVLTDPEAAKYVHGIAVHWYLDFLAPAVATLGETHRLFPNTMLFASEACVGSKFWEQSVRLGSWDRGMQYSHSIITNLLYHVVGWTDWNLALNPEGGPNWVRNFVDSPIIVDITKDTFYKQPMFYHLGHFSKFIPEGSQRVGLVASQKNDLDAVALMHPDGSAVVVVLNRSSKDVPLTIKDPAVGFLETISPGYSI HTYLWRRQSEQ ID NO: 13 ARPCIPKSFGYSSVVCVCNATYCDSFDPPTFPALGTFSRYESTRSGRRMELSMGPIQANHTGTGLLLTLQPEQKFQKVKGFGGAMTDAAALNILALSPPAQNLLLKSYFSEEGIGYNIIRVPMASCDFSIRTYTYADTPDDFQLHNFSLPEEDTKLKIPLIHRALQLAQRPVSLLASPWTSPTWLKTNGAVNGKGSLKGQPGDIYHQTWARYFVKFLDAYAEHKLQFWAVTAENEPSAGLLSGYPFQCLGFTPEHQRDFIARDLGPTLANSTHHNVRLLMLDDQRLLLPHWAKVVLTDPEAAKYVHGIAVHWYLDAFAPAAATLGETHRLFPNTMLFASEACVGSKFWEQSVRLGSWDRGMQYSHSIITNLLYHVVGWTDWNLALNPEGGPNWVRNFVDSPIIVDITKDTFYKQPMFYHLGHFSKFIPEGSQRVGLVASQKNDLDAVALMHPDGSAVVVVLNRSSKDVPLTIKDPAVGFLETISPGYSI HTYLWRRQSEQ ID NO: 14 ARPCIPKSFGYSSVVCVCNATYCDSFDPPTFPALGTFSRYESTRSGRRMELSMGPIQANHTGTGLLLTLQPEQKFQKVKGFGGAMTDAAALNILALSPPAQNLLLKSYFSEEGIGYNIIRVPMASCDFSIRTYTYADTPDDFQLHNFSLPEEDTKLKIPLIHRALQLAQRPVSLLASPWTSPTWLKTNGAVNGKGSLKGQPGDIYHQTWARYFVKFLDAYAEHKLQFWAVTAENEPSAGLLSGYPFQCLGFTPEHQRDFIARDLGPTLANSTHHNVRLLMLDDQRLLLPHWAKVVLTDPEAAKYVHGIAVHWYLDAFAPAVATLGETHRLFPNTMLFASEACVGSKFWEQSVRLGSWDRGMQYSHSIITNLLYHVVGWTDWNLALNPEGGPNWVRNFVDSPIIVDITKDTFYKQPMFYHLGHFSKFIPEGSQRVGLVASQKNDLDAVALMHPDGSAVVVVLNRSSKDVPLTIKDPAVGFLETISPGYSI HTYLWRRQSEQ ID NO: 15 ARPCIPKSFGYSSVVCVCNATYCDSFDPPTFPALGTFSRYESTRSGRRMELSMGPIQANHTGTGLLLTLQPEQKFQKVKGFGGAMTDAAALNILALSPPAQNLLLKSYFSEEGIGYNIIRVPMASCDFSIRTYTYADTPDDFQLLNFSLPEEDTKLKIPLIHRALQLAQRPVSLLASPWTSPTWLKTNGAVNGKGSLKGQPGDIYHQTWARYFVKFLDAYAEHKLQFWAVTAENEPSAGLLSGYPFQCLGFTPEHQRDFIARDLGPTLANSTHHNVRLLMLDDQRLLLPHWAKVVLTDPEAAKYVHGIAVHWYLDFLAPAAATLGETHRLFPNTMLFASEACVGSKFWEQSVRLGSWDRGMQYSHSIITNLLYHVVGWTDWNLALNPEGGPNWVRNFVDSPIIVDITKDTFYKQPMFYHLGHFSKFIPEGSQRVGLVASQKNDLDAVALMHPDGSAVVVVLNRSSKDVPLTIKDPAVGFLETISPGYSI HTYLWRRQSEQ ID NO: 16 ARPCIPKSFGYSSVVCVCNATYCDSFDPPTFPALGTFSRYESTRSGRRMELSMGPIQANHTGTGLLLTLQPEQKFQKVKGFGGAMTDAAALNILALSPPAQNLLLKSYFSEEGIGYNIIRVPMASCDFSIRTYTYADTPDDFQLLNFSLPEEDTKLKIPLIHRALQLAQRPVSLLASPWTSPTWLKTNGAVNGKGSLKGQPGDIYHQTWARYFVKFLDAYAEHKLQFWAVTAENEPSAGLLSGYPFQCLGFTPEHQRDFIARDLGPTLANSTHHNVRLLMLDDQRLLLPHWAKVVLTDPEAAKYVHGIAVHWYLDFLAPAVATLGETHRLFPNTMLFASEACVGSKFWEQSVRLGSWDRGMQYSHSIITNLLYHVVGWTDWNLALNPEGGPNWVRNFVDSPIIVDITKDTFYKQPMFYHLGHFSKFIPEGSQRVGLVASQKNDLDAVALMHPDGSAVVVVLNRSSKDVPLTIKDPAVGFLETISPGYSI HTYLWRRQ

1. A variant, recombinant β-glucocerebrosidase protein, characterized ashaving increased stability relative to wild-type, recombinantβ-glucocerebrosidase. 2-4. (canceled)
 5. The variant, recombinantβ-glucocerebrosidase protein of claim 1 wherein the increased stabilityis monitored at conditions of about neutral pH and about 37° C. over aperiod of about three hours.
 6. (canceled)
 7. The variant, recombinantβ-glucocerebrosidase protein of claim 1 wherein the increased stabilityis inside cells. 8-9. (canceled)
 10. The variant, recombinantβ-glucocerebrosidase protein of claim 1 wherein the increased stabilityis characterized by reduced proteolytic degradation. 11-13. (canceled)14. The variant, recombinant β-glucocerebrosidase protein of claim 1wherein the increased stability is in buffer solutions during proteinpurification. 15-25. (canceled)
 26. A variant, recombinantβ-glucocerebrosidase protein, characterized as retaining more catalyticactivity relative to wild-type, recombinant β-glucocerebrosidase. 27-28.(canceled)
 29. The variant, recombinant β-glucocerebrosidase protein ofclaim 26 wherein more catalytic activity is retained at conditions ofabout neutral pH and about 37° C. 30-31. (canceled)
 32. The variant,recombinant β-glucocerebrosidase protein of claim 26 wherein morecatalytic activity is retained inside cells. 33-50. (canceled)
 51. Avariant, recombinant β-glucocerebrosidase protein, comprising an aminoacid variation at one or more of the following positions: 316, 317, 321and
 145. 52. The variant, recombinant β-glucocerebrosidase proteinaccording to claim 51, further comprising one of the amino acidvariations: F316A and L317F; K321N; K321A; K321A; H145L; F316A, L317F,and K321N; F316A, L317F, and K321A; F316A, L317F, and K321V; H145L andK321N; H145L and K321A; H145L and K321V; H145L, F316A, and L317F.53-159. (canceled)
 160. A method of making a variant, recombinant3-glucocerebrosidase protein having increased catalytic activityrelative to wild-type, recombinant 3-glucocerebrosidase, the methodcomprising: (a) expressing the variant, recombinant 3-glucocerebrosidaseprotein; and (b) purifying the variant, recombinant 3-glucocerebrosidaseprotein.
 161. A composition comprising the variant, recombinantβ-glucocerebrosidase protein of any one of claims 51 or 52 and apharmaceutically acceptable carrier.
 162. A composition comprising thevariant, recombinant β-glucocerebrosidase protein of any one of claims51 or 52 and a pharmaceutically acceptable buffer.
 163. A variant,recombinant β-glucocerebrosidase protein, comprising one or morereplacement amino acids in the loop1 region of the protein, the one ormore replacement amino acids characterized as having a side-chainconformation that increases order near the active site of the protein.164-167. (canceled)
 168. A variant, recombinant β-glucocerebrosidaseprotein, comprising one or more replacement amino acids in the α-helixnear the active site (α6) of the protein, the one or more replacementamino acid sides characterized as having a side-chain confirmation thatstabilizes this helix and pulls adjacent loop1 away from the catalyticsite and has an open and active conformation. 169-172. (canceled)
 173. Avariant, recombinant β-glucocerebrosidase protein, comprising one ormore replacement amino acids in the random coil region betweenbeta-sheet (β2) and an α-helix (α2), the one or more replacement aminoacid side-chains characterized as having a side-chain confirmation thatfacilitates better interactions between different residues and secondarystructures for improved stability. 174-177. (canceled)
 178. A method fortreating a lysosomal storage disease, comprising administering to asubject in need thereof a variant, recombinant β-glucocerebrosidaseprotein characterized as having increased stability compared towild-type, recombinant β-glucocerebrosidase.
 179. A method for treatinga lysosomal storage disease, comprising administering to a subject inneed thereof a variant, recombinant β-glucocerebrosidase proteincharacterized as retaining more catalytic activity compared towild-type, recombinant β-glucocerebrosidase.
 180. A method for treatinga lysosomal storage disease, comprising administering to a subject inneed thereof a variant, recombinant β-glucocerebrosidase proteincharacterized as having an increased specific activity compared towild-type, recombinant β-glucocerebrosidase.
 181. A method for treatinga lysosomal storage disease, comprising administering to a subject inneed thereof a variant, recombinant β-glucocerebrosidase proteincomprising an amino acid variation at one or more of the followingpositions: 316, 317, 321 and
 145. 182. The method for treating alysosomal storage disease of claim 181, wherein the variant, recombinantβ-glucocerebrosidase protein further comprises one of the followingamino acid variations: F316A and L317F; K321N; K321A; K321V; H145L;H145F; F316A, L317F, and K321N; F316A, L317F, and K321A; F316A, L317F,and K321V; H145L, F316A, and L317F; H145L and K321N; H145L and K321A;H145L and K321V; H145L, F316A, L317F and K321N; H145L, F316A, L317F andK321A; H145L, F316A, L317F and K321V. 183-197. (canceled)
 198. Themethod of claim 182 wherein the lysosomal storage disease is Gaucherdisease.
 199. The method of claim 198, wherein the administration isintravenous infusion.
 200. The method of claim 198, wherein theadministration is intraperitoneal injection.
 201. A compound comprisinga variant, recombinant β-glucocerebrosidase protein characterized ashaving any one of the following amino acid sequences: SEQ ID NO: 2, SEQID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ IDNO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ IDNO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO:
 16. 202. A compositioncomprising a variant, recombinant β-glucocerebrosidase proteincharacterized as having any one of the following amino acid sequences:SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6,SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11,SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ IDNO:
 16. 203. A variant, recombinant β-glucocerebrosidase proteincharacterized as capable of increased expression relative to wild-typeβ-glucocerebrosidase. 204-211. (canceled)
 212. A method of making acompound comprising a variant, recombinant β-glucocerebrosidase proteincharacterized as having any one of the following amino acid sequences:SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6,SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11,SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ IDNO:
 16. 213. A method of making a nucleic acid encoding a variant,recombinant β-glucocerebrosidase protein characterized as having any oneof the following amino acid sequences: SEQ ID NO: 2, SEQ ID NO: 3, SEQID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ IDNO: 9, SEQ ID NO: 10, or SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13,SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16.